Carboline derivatives useful in the inhibition of angiogenesis

ABSTRACT

In accordance with the present invention, compounds that inhibit the expression of VEGF post-transcriptionally have been identified, and methods for their use provided. In one aspect of the invention, compounds useful in the inhibition of VEGF production, in the treatment of solid tumor cancer, and in reducing plasma and/or tumor VEGF levels, are provided. In another aspect of the invention, methods are provided for the inhibition of VEGF production, the treatment of cancer, and the reduction of plasma and/or tumor VEGF levels, using the compounds of the invention.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.11/107,783, filed Apr. 18, 2005, currently allowed, which is acontinuation-in-part of U.S. application Ser. No. 11/079,420, filed Mar.15, 2005, now U.S. Pat. No. 7,601,840, issued Oct. 13, 2009, whichclaims the benefit of and priority to U.S. Provisional Application No.60/552,725, filed Mar. 15, 2004, which applications are incorporatedherein by reference in their entireties. This application also claimspriority to International Application No. PCT/US2005/08481, filed Mar.15, 2005, which application is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Federal Award ID1R43CA108330-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods and compounds for inhibitingangiogenesis. More particularly, the present invention relates tomethods and compounds for inhibiting angiogenesis.

BACKGROUND OF THE INVENTION

Aberrant angiogenesis plays a critical role in the pathogenesis ofnumerous diseases, including malignant, ischemic, inflammatory andimmune disorders (Carmeliet, Nat. Med., 9(6):653-60 (2003), Ferrara,Semin. Oncol., 29(6 Suppl 16):10-4 (2002)). The best-known of thesedisorders are cancer, exudative macular degeneration and diabeticretinopathy (DR), the last two of which are leading cause of blindnessin the United States (Witmer et al., Prog. Retin Eye Res., 22(1):1-29(2003), Clark et al., Nat. Rev. Drug Discovery, 2:448-459 (2003)).During the last decade our understanding of the molecular basis ofangiogenesis has grown considerably. Numerous cytokines and growthfactors that stimulate angiogenesis, such as VEGF, FGF-2, PDGF, IGF-1,TGF, TNF-α, G-CSF have been identified (Ferrara et al., Nat. Med.,5(12):1359-64 (1999), Kerbel et al., Nat. Rev. Cancer, 2(10):727-39(2002), Rofstad et al., Cancer Res., 60(17):4932-8 (2000)). Among thesegrowth factors, Vascular Endothelial Growth Factor (VEGF) plays acentral role in angiogenesis (Ferrara, Semin. Oncol., 29(6 Suppl 16):10-4 (2002)).

VEGF, also known as VEGF-A, was initially identified for its ability toinduce vascular permeability and to promote vascular endothelial cellproliferation (Leung et al., Science, 246:1306-1309 (1989), Plouet etal., EMBO J., 8:3801-3806 (1989), Connolly et al., J. Biol. Chem.,264:20017-20024 (1989)). VEGF is encoded by a single gene that givesrise to four isoforms by alternative splicing (Tischer et al., J. Biol.Chem., 266:11947-11954 (1991)). All four isoforms share the sameunusually long and GC rich 5′-UTR, as well as a 3′-UTR that includesmultiple RNA stability determinants. The receptors VEGFR-2 (also knownas KDR or Flk-1) and VEGFR-1 (previously known as Flt1) recognize thedimeric form of VEGF (Ortega et al., Front. Biosci., 4:D141-52 (1999),Sato et al., Annals of New York Academy of Science, 902:201-207,(2000)). The highly specific VEGFR-2 receptor is expressed onendothelial cells. VEGF binding to the VEGFR-2 receptor activates thereceptor's tyrosine kinase activity, leading to endothelial cellproliferation, differentiation and primitive vessel formation (Shalabyet al., Nature, 376:62-66, (1995)). VEGFR-1 inhibits endothelial cellgrowth either by acting as a decoy or by suppressing signaling pathwaysthrough VEGFR-2 (Fong et al., Nature, 376:66-70 (1995)).

Over 30 years ago, it was proposed that inhibition of tumor angiogenesiscould be an effective approach for the treatment of cancer (Folkman, N.Engl. J. Med., 285(21):1182-6 (1971)). VEGF and its receptor have beendemonstrated to have a central role in tumor angiogenesis, especially inthe early stages of tumor growth (Hanahan et al., Cell, 86:353-364,1996)). Indeed, increased levels of VEGF expression have been correlatedwith microvessel density in primary tumor tissues (Gasparini et al., J.Natl. Cancer Inst., 89:139-147 (1997)). Moreover, increased levels ofthe VEGF transcript are found in virtually all of the common solidtumors (Ferrara et al., Endocr. Rev., 18:4-25, 1997)). In general,tumor-bearing patients have higher levels of VEGF compared to those intumor-free individuals, and high VEGF levels in serum/plasma areassociated with poor prognosis (Dirix et al., Br. J. Cancer, 76:238-243(1997)). Consistent with the role of VEGF in tumor angiogenesis, VEGFnull embryonic stem cells showed a dramatically reduced ability to formtumors in nude mice (Carmeliet et al., Nature, 380:435-439 (1996)).Direct evidence for the involvement of VEGF in tumorgenesis wasdemonstrated by using specific antibodies against VEGF in humanxenografts implanted in nude mice (Kim et al., Nature, 362:841-844(1993), Hichlin et al., Drug Discovery Today, 6:517-528 (2001)). Inthese studies, the inhibition of tumor growth correlated positively withdecreased vessel formation in the antibody-treated tumors. Subsequentexperiments using the soluble receptors substantiated the importance ofVEGF activity in tumor growth (Lin et al., Cell Growth Differ.,9(1):49-58 (1998)), and demonstrated that inactivation of VEGF byspecific antibody treatment directly resulted in a nearly completesuppression of tumor-associated neovascularization (Borgstrom et al.,Prostate, 35:1-10 (1998), Yuan et al., Proc. Natl. Acad. Sci. USA,93:14765-14770 (1996)).

In exudative macular degeneration and diabetic retinopathy, pre-clinicalexperiments and clinical trials have demonstrated that over productionof VEGF is critical for aberrant retinal or choroidal neovascularization(reviewed in Witmer et al., Prog. Retin Eye Res., 22(1):1-29 (2003)).Evidence has been obtained that intra-ocular VEGF levels are stronglycorrelated with active retinal/choroidal neovascularization (CNV) inpatients with diseases such as diabetic retinopathy and wet form maculardegeneration (Funatsu et al., Am. J. Ophthalmol., 133(4):537-43 (2002),Lip et al., Ophthalmology, 108(4):705-10 (2001)). In addition, studiesusing transgenic mice demonstrated that overexpression of VEGF inretinal pigment epithelial cells or photoreceptor cells results inchoroidal or retinal neovasucularization (Schwesinger et al., Am. J.Pathol., 158(3):1161-72 (2001), Ohno-Matsui et al., Am. J. Pathol.,160(2):711-9 (2002)). In recent studies neutralizing antibodies, solublereceptor, receptor antagonists, or siRNA have proven efficacious inreducing VEGF-mediated blood vessel formation in animal models and inthe clinic. (Eyetech Study Group, 22(2):143-52 (2002), Krzystolik etal., Arch. Ophthalmol., 120(3):338-46 (2002), Shen et al., Lab Invest.,82(2):167-82 (2002), Honda et al., Gene Ther., 7(11):978-85 (2000),Saishin et al., J. Cell Physiol., 195(2):241-8 (2003)).

VEGF expression is regulated by a number of factors and agents includingcytokines, growth factors, steroid hormones and chemicals, and mutationsthat modulate the activity of oncogenes such as ras or the tumorsuppressor gene VHL (Maxwell et al., Nature, 399:271-275 (1999), Rak etal., Cancer Res., 60:490-498 (2000)). Nevertheless, hypoxia is the mostsignificant physiologic signal for regulating VEGF expression. Hypoxiaresults in enhanced VEGF expression by increasing both the transcriptionrate and stability of the VEGF transcript (Ikeda et al., J. Biol. Chem.270:19761-19766 (1995), Stein et al., Mol. Cell. Biol. 18:3112-3119(1998), Levy et al., J. Biol. Chem. 271:2746-2753 (1996)).Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor thatincreases VEGF gene expression in cells undergoing hypoxia by binding tothe hypoxia response element (HRE) located in the VEGF promoter (Liu etal., Circ. Res., 77:638-643 (1995), Semenza, Annu. Rev. Cell. Dev.Biol., 5:551-578 (1999)). Both the stability and translation efficiencyof the VEGF transcript is influenced by sequences in the 5′- and3′-untranslated regions (UTRs). The 5′-UTR contains an internalribosomal entry site (IRES) and mediates cap-independent translationinitiation while the 3′-UTR harbors multiple AU-rich (AUR) stabilitydeterminants that have been previously shown to regulate turnover ofVEGF mRNA. In addition, the translation initiation of the VEGFtranscript is uniquely regulated. Under hypoxic conditions, translationof most cellular transcripts mediated by cap-dependent translationinitiation process is greatly impaired (Kraggerud et al., AnticancerRes., 15:683-686 (1995)). Initiation of translation of the VEGF mRNA,however, is unique under hypoxic conditions in that it is mediated viaan internal ribosome entry site (IRES) within the VEGF 5′UTR (Stein etal., Mol. Cell. Biol. 18:3112-3119 (1998), Levy et al., J. Biol. Chem.271:2746-2753 (1996), Huez et al., Mol. Cell. Biol., 18:6178-6190(1998), Akiri et al., Oncogene, 17:227-236 (1998)). Thus, this form ofpost-transciptional regulation permits cells to produce large amounts ofVEGF protein to support either further tumor growth or aberrantneovascularization in ocular diseases under hypoxic conditions. Thestability of VEGF mRNA is also greatly enhanced as a consequence of thebinding of factors to elements in the 3′-UTR (Goldberg et al., J. Biol.Cell. J. Biol. Chem., 277(16):13635-40 (2002)).

There is a large body of experimental evidence indicating that tumorgrowth can be inhibited by the prevention of neovascularization (Lin etal., Cell Growth Differ., 9(1):49-58 (1998), Zhu et al., Invest. NewDrugs, 17:195-212 (1999)). Tumor vessels are generally immature andconstantly undergo remodeling (Carmeliet, Nat. Med., 9(6):653-60 (2003),Carmeliet et al., Nature, 407:249-257 (2000)). Active and aberrantangiogenesis is the result of a disruption in the normal balance ofproangiogenic and anti-angiogenic factors, including various cytokines,growth factors and steroid hormones. Despite the complexity of theregulation of tumor angiogenesis, accumulated evidence indicates thattargeting a single proangiogenic factor might be sufficient to inhibittumor angiogenesis and suppress tumor growth (Kim et al., Nature,362:841-844 (1993), Millauer et al., Nature, 367:576-579 (1994), Fong etal., Cancer Res., 59:99-106 (1999)). Among many angiogenesis targets,VEGF and its receptor are most attractive (Carmeliet, Nat. Med.,9(6):653-60 (2003), Ortega et al., Front. Biosci., 4:D141-52 (1999)). Asnoted above, treatment with a monoclonal antibody specifically targetingVEGF inhibited the growth of tumors in human xenografts implanted innude mice. Subsequently, various approaches designed to inactivate VEGFsignaling have been tested in tumor models and have proven to be highlyeffective in a broad range of tumor cell lines including carcinomas,sarcomas and gliomas (Ferrara et al., Endocr. Rev., 18:4-25, 1997), Kimet al., Nature, 362:841-844 (1993), Millauer et al., Nature, 367:576-579(1994), Fong et al., Cancer Res., 59:99-106 (1999), Geng et al., CancerRes., 61:2413-2419 (2001)). In addition, inhibition of VEGF by anti-VEGFantibody did not result in significant side effects in fully developedrodents or primates (Ryan et al, Toxicol. Pathol., 27:78-86 (1999),Ferrara et al., Nat. Med., 4:336-340 (1998)). Taken together, theseresults indicate that VEGF is a valid target for the development oftumor therapy. Indeed, a number of clinical trials are underway usingVEGF inhibitors (Matter, Drug Discovery Today, 6:1005-1024 (2001),Hichlin et al., Drug Discovery Today, 6:517-528 (2001)).

Although several pro-angiogenic factors are implicated in the pathologyof exudative age-related macular degeneration, VEGF appears to be themost critical in the pathogenesis and development of this disease(Witmer et al., Prog. Retin Eye Res., 22(1):1-29 (2003), Holash et al.,Science, 284:1994-1998 (1999)). Data from preclinical experiments andclinical trials have demonstrated that blockade of VEGF alone issufficient to alleviate or stabilize disease progression (Eyetech StudyGroup, 22(2):143-52 (2002), Krzystolik et al., Arch. Ophthalmol.,120(3):338-46 (2002), Shen et al., Lab Invest., 82(2):167-82 (2002),Honda et al., Gene Ther., 7(11):978-85 (2000), Saishin et al., J. CellPhysiol., 195(2):241-8 (2003)). For example, inhibition of VEGFRsignaling by a specific tyrosine kinase inhibitor is sufficient tocompletely prevent retinal neovascularization in a murine retinopathy ofprematurity model (Ozaki H, Seo M S, Ozaki et al., Am. J. Pathol.,156(2):697-707 (2000)). Furthermore, it has recently been demonstratedthat small interfering RNAs (siRNA) directed against murine VEGFsignificantly inhibited ocular neovascularization after laserphotocoagulation in a mouse model (Reich et al., Mol. Vis. 30;9:210-6(2003)). These results indicate that selective inhibition of VEGFexpression is achievable and offers validation of this approach for thetreatment of ocular neovascular diseases such as exudative maculardegeneration and diabetic retinopathy.

Three approaches have been used to inhibit VEGF activity, including (1)neutralization of VEGF activity by using a specific antibody, solubleVEGF receptor or aptamer oligos against the VEGF/VEGFR interaction (Kimet al., Nature, 362:841-844 (1993), Lin et al., Cell Growth Differ.,9(1):49-58 (1998), Borgstrom et al., Prostate, 35:1-10 (1998), Zhu etal., Invest. New Drugs, 17:195-212 (1999), Millauer et al., Nature,367:576-579 (1994), Asano et al., Jpn. J. Cancer Res., 90(1):93-100(1999), Brekken et al., Cancer Res., 60(18):5117-24 (2000)); (2)inhibition of VEGFR mediated signal transduction by specific smallmolecule tyrosine kinase inhibitors (Fong et al., Cancer Res., 59:99-106(1999), Wedge et al., Cancer Res., 60(4):970-5 (2000), Laird et al.,Cancer Res., 60(15):4152-60 (2000)); and (3) inhibition of VEGF/VEGFRexpression by using antisense, siRNA or ribozyme (Reich et al., Mol.Vis. 30;9:210-6 (2003), Parry et al., Nucleic Acids Res., 27:2569-2577(1999), Ellis et al., Surgery, 120:871-878 (1996), Filleur et al.,Cancer Res., 63(14):3919-22 (2003)). Although all of these approachesshow significant inhibition of angiogenesis in vivo, they all possesssignificant limitations. For example, therapeutic proteins (antibody andsoluble receptors) or oligos (antisense, siRNA and ribozyme) are largemolecules with poor permeability that usually require parenteraladministration and are costly to produce. For treatment of chronicocular neovascularization, multiple injections may be impractical due topotential complications such as retinal detachment and procedure relatedinfection. Moreover, tyrosine kinase inhibitors have the potential forlimited specificity. VEGF is constitutively expressed at a low level innormal eyes and other tissues and thus it may be harmful to completelysuppress VEGF function by administration of antibody or tyrosine kinaseinhibitors systemically, especially for patients with AMD and RD many ofwhom are also hypertensive (Giles et al., Cancer, 97(8):1920-8 (2003),Sugimoto et al., J. Biol. Chem., 278(15):12605-8 (2003), Bergsland etal., American Society of Clinical Oncology 36^(th) Annual Meeting, 20-23May, 2000, New Orleans, La., USA, Abstract 939), DeVore et al., AmericanSociety of Clinical Oncology 36^(th) Annual Meeting, 20-23 May, 2000,New Orleans, La., USA, Abstract 1896).

Thus, there remains a need to develop, characterize and optimize leadmolecules for the development of novel anti-angiogenesis drugs.Accordingly, it is an object of the present invention to provide suchcompounds.

All documents referred to herein are incorporated by reference into thepresent application as though fully set forth herein.

SUMMARY OF THE INVENTION

In accordance with the present invention, compounds that inhibit theexpression of VEGF post-transcriptionally have been identified, andmethods for their use provided.

In one aspect of the invention, compounds of Formulas (I), (II) and(III), including Formulas (I-a) to (I-l), are provided which are usefulin the inhibition of VEGF production, in the inhibition of angiogenesis,and/or in the treatment of cancer, diabetic retinopathy or exudativemacular degeneration.

In another aspect of the invention, methods are provided for theinhibition of VEGF production, the inhibition of angiogenesis, and/orthe treatment of cancer, diabetic retinopathy, rheumatoid arthritis,psoriasis, atherosclerosis, chronic inflammation, other chronicinflammation-related diseases and disorders, obesity, or exudativemacular degeneration using the compounds described herein.

In one embodiment, the invention is directed to methods for inhibitingVEGF production comprising administering a VEGF-expression inhibitingamount of at least one compound of the invention to a subject in needthereof.

In another embodiment, methods for inhibiting angiogenesis are providedcomprising administering an anti-angiogenic amount of at least onecompound of the invention to a subject in need thereof.

The present invention also provides methods for treating a solid tumorcancer comprising administering a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

Solid tumor cancers that can be treated by the present invention includesolid tumor carcinomas and solid tumor sarcomas. Solid tumor carcinomasinclude, but are not limited to, pediatric solid tumors, such as Wilmstumor and neuroblastoma, and carcinomas of the epidermis, such asmalignant melanomas, as well as lung cancers, cervical cancers, coloncancers and renal cancers. Solid tumor sarcomas include, but are notlimited to, fibrosarcomas.

The present invention also provides methods for treating a Wilms tumorcomprising administering a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention also provides methods for treating a neuroblastomacomprising administering a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention further provides methods of treating a malignantmelanoma comprising administering a therapeutically effective amount ofa compound of Formula (I), or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention yet further provides methods for treating cervicalcancer comprising administering a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention also provides methods for treating lung cancercomprising administering a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention also provides methods for treating colon cancercomprising administering a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention further provides methods for treating a solidtumor cancer by post-transcriptionally modifying VEGF expressioncomprising administering a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention yet further provides methods for treating a solidtumor cancer by slowing tumorigenesis at a pre-vascular stage comprisingadministering a therapeutically effective amount of a compound ofFormula (I), or a pharmaceutically acceptable salt, hydrate, solvate,clathrate, polymorph, racemate or stereoisomer of said compound, to asubject in need thereof.

The present invention also provides methods for treating a solid tumorcancer by reducing tumor VEGF levels comprising administering atherapeutically effective amount of a compound of Formula (I), or apharmaceutically acceptable salt, hydrate, solvate, clathrate,polymorph, racemate or stereoisomer of said compound, to a subject inneed thereof.

The present invention further provides methods for treating a solidtumor cancer by reducing perivascularly sequestered VEGF comprisingadministering a therapeutically effective amount of a compound ofFormula (I), or a pharmaceutically acceptable salt, hydrate, solvate,clathrate, polymorph, racemate or stereoisomer of said compound, to asubject in need thereof.

The present invention also provides methods of blocking VEGF mRNAtranslation comprising administering a therapeutically effective amountof a compound of Formula (I), or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention further provides methods of slowing tumorigenesisat a pre-vascular stage comprising administering a therapeuticallyeffective amount of a compound of Formula (I), or a pharmaceuticallyacceptable salt, hydrate, solvate, clathrate, polymorph, racemate orstereoisomer of said compound, to a subject in need thereof.

The present invention yet further provides methods of reducing tumorVEGF levels comprising administering a therapeutically effective amountof a compound of Formula (I), or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention also provides methods of reducing plasma VEGFlevels comprising administering a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, polymorph, racemate or stereoisomer of saidcompound, to a subject in need thereof.

The present invention further provides methods of reducingperivascularly sequestered VEGF comprising administering atherapeutically effective amount of a compound of Formula (I), or apharmaceutically acceptable salt, hydrate, solvate, clathrate,polymorph, racemate or stereoisomer of said compound, to a subject inneed thereof.

The present invention yet further provides methods of treating a solidtumor cancer comprising measuring plasma levels of VEGF, tumor levels ofVEGF, or both, and administering administering a therapeuticallyeffective amount of a compound of Formula (I), or a pharmaceuticallyacceptable salt, hydrate, solvate, clathrate, polymorph, racemate orstereoisomer of said compound, to a subject in need thereof.

The present invention also provides methods of diagnosing solid tumorcancers comprising measuring plasma levels of VEGF.

The present invention further provides methods of diagnosing solid tumorcancers comprising measuring tumor levels of VEGF.

The present invention yet further provides methods of treating a solidtumor cancer comprising administering a therapeutically effective amountof a compound of Formula (I), or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, polymorph, racemate or stereoisomerthereof, together with one or more additional cancer agents to a subjectin need thereof.

These and other aspects of the invention will be more clearly understoodwith reference to the following preferred embodiments and detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates inhibition of VEGF expression in a cervical cancertumor by a certain compound of the invention.

FIG. 2 illustrates inhibition of lung cancer tumor growth by a certaincompound of the invention.

FIG. 3 illustrates inhibition of Wilms and neuroblastoma tumor growth bya certain compound of the invention.

FIG. 4 illustrates inhibition of fibrosarcoma tumor growth by a certaincompound of the invention.

FIG. 5 illustrates inhibition of malignant melanoma tumor growth by acertain compound of the invention.

FIG. 6 illustrates inhibition of VEGF expression in a fibrosarcoma tumorby a certain compound of the invention.

FIG. 7 illustrates inhibition of colon cancer tumor growth by a certaincompound of the invention, either alone or in combination with 5-FU(fluorouracil).

FIG. 8 illustrates inhibition of cervical cancer tumor growth by acertain compound of the invention, either alone or in combination withTaxol (paclitaxel).

FIG. 9 illustrates inhibition of fibrosarcoma tumor growth by a certaincompound of the invention, either alone or in combination with Avastin(bevacizumab, an antibody to VEGF).

FIG. 10 illustrates inhibition of tumor and plasma VEGF expression in afibrosarcoma tumor by a certain compound of the invention, either aloneor in combination with Avastin.

FIG. 11 illustrates inhibition of angiogenesis by a certain compound ofthe invention.

FIG. 12 illustrates that a certain compound of the invention does notaffect VEGF mRNA levels under hypoxic conditions.

FIG. 13 illustrates that the activity of phosphdiesterase 5 (PDE-5) isnot affected by certain compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Aberrant up-regulation of Vascular Endothelial Growth Factor (VEGF), akey factor for angiogenesis, is an important contributor to thepathogenesis of disease states such as cancer, diabetic retinopathy,rheumatoid arthritis, psoriasis, atherosclerosis, chronic inflammation,other chronic inflammation-related diseases and disorders, obesity, orexudative macular degeneration. In accordance with the presentinvention, compounds that inhibit the expression of VEGFpost-transcriptionally have been identified, and methods for their useprovided. The compounds of the invention have nanomolar to sub-nanomolaractivity for the inhibition of VEGF expression.

A. Compounds of the Invention

In one aspect of the invention, compounds are provided which are usefulin the inhibition of VEGF production, in the inhibition of angiogenesis,and/or in the treatment of cancer, diabetic retinopathy or exudativemacular degeneration. In certain embodiments, the compounds of theinvention specifically inhibit VEGF production, while in otherembodiments, the compounds of the invention inhibit VEGF expression aswell as that of other angiogenesis factors such as FGF-2. In thisregard, pan-angiogenic inhibitor may be preferred in methods ofinhibiting tumor growth, while VEGF specific inhibitors may be preferredfor the treatment of ocular neovascular disorders (Eyetech Study Group,22(2):143-52 (2002)).

The compounds of the invention generally include one or more chiralcenters, and as such may exist as racemic mixtures (R/S) or asenantiomerically pure compositions. The compounds may exist as (R) or(S) isomers (when one chiral center is present) in enantiomerically purecompositions. In a preferred embodiment, the compounds of the inventionare the (S) isomers and may exist as enantiomerically pure compositionscomprising only the (S) isomer. As one of skill will recognize, whenmore than one chiral center is present, the compounds of the inventionmay exist as (R,R), (R,S), (S,R), (S,S), etc. isomer. Preferredcompounds included (S,S) and (S,R) isomers.

As used herein, “racemic mixture” refers to compositions consistingsubstantially of greater than 50%, 70%, 90%, or 99% of one of theisomers.

As used herein, “enantiomerically pure” refers to compositionsconsisting substantially of a single isomer, preferably consisting of90%, 92%, 95%, 98%, 99%, or 100% of a single isomer.

Preferred compounds of the present invention useful in the inhibition ofVEGF production include those of Formula (I) as shown below.

wherein,

X is hydrogen; a C₁ to C₆ alkyl, optionally substituted with one or morehalogens; a hydroxyl group; a halogen; a C₁ to C₅ alkoxy, optionallysubstituted with a C₆ to C₁₀ aryl group;

A is C or N;

B is C or N, with the proviso that at least one of A or B is N, and thatwhen A is N, B is C;

R₁ is a hydroxyl group; a C₁ to C₈ alkyl group, optionally substitutedwith an alkylthio group, a 5 to 10 membered heteroaryl, a C₆ to C₁₀ arylgroup optionally substituted with at least one independently selected R₀group; a C₂ to C₈ alkyenyl group; a C₂ to C₈ alkynyl group; a 3 to 12membered heterocycle group, wherein the heterocycle group is optionallysubstituted with at least one independently selected halogen, oxo,amino, alkylamino, acetamino, thio, or alkylthio group; a 5 to 12membered heteroaryl group, wherein the heteroaryl group is optionallysubstituted with at least one independently selected halogen, oxo,amino, alkylamino, acetamino, thio, or alkylthio group; or a C₆ to C₁₀aryl group, optionally substituted with at least one independentlyselected R₀ group;

R₀ is a halogen; a cyano; a nitro; a sulfonyl, wherein the sulfonyl isoptionally substituted with a C_(i) to C₆ alkyl or a 3 to 10 memberedheterocycle; an amino group, wherein the amino group is optionallysubstituted with a C₁ to C₆ alkyl, —C(O)—R_(b), —C(O)O—R_(b), asulfonyl, an alkylsulfonyl, a 3 to 10 membered heterocycle groupoptionally substituted with a —C(O)O—R_(a); —C(O)—NH—R_(b); a 5 to 6membered heterocycle; a 5 to 6 membered heteroaryl; a C₁ to C₆ alkylgroup, wherein the alkyl group is optionally substituted with at leastone independently selected hydroxyl, halogen, amino, or 3 to 12 memberedheterocycle group, wherein the amino group and heterocycle group areoptionally substituted with at least one independently selected C₁ to C₄alkyl group, which C₁ to C₄ alkyl group is optionally substituted withat least one independently selected C₁ to C₄ alkoxy group, amino group,alkylamino group, or 5 to 10 membered heterocycle group; a —C(O)—R_(n)group; or an —OR_(a) group;

R_(a) is hydrogen; C₂ to C₈ alkylene; a —C(O)O—R_(b) group; a—C(O)—NH—R_(b); a C₁ to C₈ alkyl, wherein the alkyl group is optionallysubstituted with at least one independently selected hydroxyl, halogen,C₁ to C₄ alkoxy, amino, alkylamino, acetamide, —C(O)—R_(b),—C(O)O—R_(b), C₆ to C₁₀ aryl, 3 to 12 membered heterocycle, or 5 to 12heteroaryl group, further wherein the alkylamino is optionallysubstituted with a hydroxyl, a C₁ to C₄ alkoxy, or a 5 to 12 memberedheteroaryl optionally substituted with a C₁ to C₄ alkyl, further whereinthe acetamide is optionally substituted with a C_(i) to C₄ alkoxy,sulfonyl, or alkylsulfonyl, further wherein and the heterocycle group isoptionally substituted with a C_(i) to C₄ alkyl optionally substitutedwith a hydroxyl group, —C(O)—R_(n), —C(O)O—R_(n), or an oxo group;

R_(b) is hydroxyl; an amino; an alkylamino, wherein the alkylamino isoptionally substituted with a hydroxyl, an amino, an alkylamino, a C₁ toC₄ alkoxy, a 3 to 12 membered heterocycle optionally substituted with atleast one independently selected C₁ to C₆ alkyl, oxo, —C(O)O—R_(a), or a5 to 12 membered heteroaryl optionally substituted with a C₁ to C₄alkyl; a C₁ to C₄ alkoxy; a C₂ to C₈ alkenyl; a C₂ to C₈ alkynyl; a C₆to C₁₀ aryl, wherein the aryl is optionally substituted with at leastone independently selected halogen or C_(i) to C₄ alkoxy; a 5 to 12membered heteroaryl; 3 to 12 membered heterocycle group, wherein theheterocycle is optionally substituted with at least one independentlyselected acetamide, —C(O)O—R_(n), 5 to 6 membered heterocycle, or C₁ toC₆ alkyl optionally substituted with a hydroxyl, C₁ to C₄ alkoxy, aminogroup, or alkylamino group; or a C₁ to C₈ alkyl, wherein the alkyl isoptionally substituted with at least one independently selected C₁ to C₄alkoxy, C₆ to C₁₀ aryl, amino, or 3 to 12 membered heterocycle group,wherein the amino and heterocycle groups are optionally substituted withat least one independently selected C₁ to C₆ alkyl, oxo, or —C(O)O—R_(n)group;

R₂ is a hydrogen; a hydroxyl; a 5 to 10 membered heteroaryl group; aC_(i) to C₈ alkyl group, wherein the alkyl group is optionallysubstituted with a hydroxyl, a C₁ to C₄ alkoxy, a 3 to 10 memberedheterocycle, a 5 to 10 membered heteroaryl, or C₆ to C₁₀ aryl group; a—C(O)—R_(c) group; a —C(O)O—R_(d) group; a —C(O)—N(R_(d)R_(d)) group; a—C(S)—N(R_(d)R_(d)) group; a —C(S)—O—R_(e) group; a —S(O₂)—R_(e) group;a —C(NR_(e))—S—R_(e) group; or a —C(S)—S—R_(f) group;

R_(e) is hydrogen; an amino, wherein the amino is optionally substitutedwith at least one independently selected C₁ to C₆ alkyl or C₆ to C₁₀aryl group; a C₆ to C₁₀ aryl, wherein the aryl is optionally substitutedwith at least one independently selected halogen, haloalkyl, hydroxyl,C₁ to C₄ alkoxy, or C₁ to C₆ alkyl group; —C(O)—R_(n); a 5 to 6 memberedheterocycle, wherein the heterocycle is optionally substituted with a—C(O)—R_(n) group; a 5 to 6 membered heteroaryl; a thiazoleamino group;a C₁ to C₈ alkyl group, wherein the alkyl group is optionallysubstituted with at least one independently selected halogen, a C₁ to C₄alkoxy, a phenyloxy, a C₆ to C₁₀ aryl, —C(O)—R_(n), —O—C(O)—R_(n),hydroxyl, or amino group, optionally substituted with a —C(O)O—R_(n)group;

R_(d) is independently hydrogen; a C₂ to C₈ alkenyl group; a C₂ to C₈alkynyl group; a C₆ to C₁₀ aryl group, wherein the aryl is optionallysubstituted with at least one independently selected halogen, nitro, C₁to C₆ alkyl, —C(O)O—R_(e), or —OR_(e); or a C₁ to C₈ alkyl group,wherein the alkyl group is optionally substituted with at least oneindependently selected halogen, C₁ to C₄ alkyl, C₁ to C₄ alkoxy,phenyloxy, C₆ to C₁₀ aryl, 5 to 6 membered heteroaryl, —C(O)—R_(n),—O—C(O)—R_(n), or hydroxyl group, wherein the C₆ to C₁₀ aryl group isoptionally substituted with at least one independently selected halogenor haloalkyl group;

R_(e) is a hydrogen; a C₁ to C₆ alkyl group, wherein the alkyl group isoptionally substituted with at least one independently selected halogenor alkoxy group; or a C₆ to C₁₀ aryl group, wherein the aryl group isoptionally substituted with at least one independently selected halogenor alkoxy group;

R_(f) is a C₁ to C₆ alkyl group, optionally substituted with at leastone independently selected halogen, hydroxyl, C₁ to C₄ alkoxy, cyano, C₆to C₁₀ aryl, or —C(O)—R_(n) group, wherein the alkoxy group may beoptionally substituted with at least one C₁ to C₄ alkoxy group and thearyl group may be optionally substituted with at least one independentlyselected halogen, hydroxyl, C₁ to C₄ alkoxy, cyano, or C₁ to C₆ alkylgroup;

R_(n) is a hydroxyl, C₁ to C₄ alkoxy, amino, or C₁ to C₆ alkyl group;

R₃ is hydrogen or —C(O)—R_(g);

R_(g) is a hydroxyl group;; an amino group, wherein the amino isoptionally substituted with a C₆ to C₁₀ cycloalkyl group or a 5 to 10membered heteroaryl group; or a 5 to 10 membered heterocycle group,wherein the heterocycle group is optionally substituted with a—C(O)—R_(n) group; and

n is 0, 1, 2, or 3.

As will be evident to one of skill in the art, the compounds of Formula(I) comprise at least one stereocenter (e.g., at the R₁ substituent),and may exist as a racemic mixture or as an enantiomerically purecomposition. In a preferred embodiment, the compounds of Formula (I) arethe (S) isomer, in an enantiomerically pure composition.

As used herein, the term “alkyl” generally refers to saturatedhydrocarbyl radicals of straight, branched or cyclic configurationincluding methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclohexyl, n-heptyl, octyl,n-octyl, and the like. In some embodiments, alkyl substituents may beinclude C₁ to C₈, C₁ to C₆, or C₁ to C₄ alkyl groups. The alkyl groupmay be optionally substituted with one or more halogen or alkoxy groups.For instance, the alkyl group may be a haloalkyl, dihaloalkyl, ortrihaloalkyl.

As used herein, “alkenyl” generally refers to linear, branched or cyclicalkene radicals having one or more carbon-carbon double bonds, such asC₂ to C₈ and C₂ to C₆ alkenyl groups, including 3-prop enyl.

As used herein, “alkynyl” generally refers to linear, branched or cyclicalkyne radicals having one or more carbon-carbon triple bonds, such asC₂ to C₈ and C₂ to C₆ alkynyl groups, including hex-3-yne.

As used herein, “aryl” refers to a carbocyclic aromatic ring structure.Included in the scope of aryl groups are aromatic rings having from fiveto twenty carbon atoms. Aryl ring structures include compounds havingone or more ring structures, such as mono-, bi-, or tricyclic compounds.Examples of aryl groups that include phenyl, tolyl, anthracenyl,fluorenyl, indenyl, azulenyl, phenanthrenyl (i.e., phenanthrene), andnapthyl (i.e., napthalene) ring structures. In certain embodiments, thearyl group may be optionally substituted.

As used herein, “heteroaryl” refers to cyclic aromatic ring structuresin which one or more atoms in the ring, the heteroatom(s), is an elementother than carbon. Heteroatoms are typically O, S or N atoms. Includedwithin the scope of heteroaryl, and independently selectable, are O, N,and S heteroaryl ring structures. The ring structure may includecompounds having one or more ring structures, such as mono-, bi-, ortricyclic compounds. In some embodiments, the heteroaryl groups may beselected from heteroaryl groups that contain one or more heteroatoms,two or more heteroatoms, three or more heteroatoms, or four or moreheteroatoms. Heteroaryl ring structures may be selected from those thatcontain five or more atoms, six or more atoms, or eight or more atoms.Examples of heteroaryl ring structures include: acridine, benzimidazole,benzoxazole, benzodioxole, benzofuran, dihydro-chromen-4-only,1,3-diazine, 1,2-diazine, 1,2-diazole, 1,4-diazanaphthalene, furan,furazan, imidazole, indole, isoxazole, isoquinoline, isothiazole,isoindolyl, oxazole, purine, pyridazine, pyrazole, pyridine, pyrazine,pyrimidine, pyrrole, quinoline, quinoxaline, thiazole, thiophene,1,3,5-triazine, 1,2,4-triazine, 1,2,3-triazine, tetrazole andquinazoline. In certain embodiments, the heteroaryl may be optionallysubstituted.

As used herein, “heterocycle” refers to cyclic ring structures in whichone or more atoms in the ring, the heteroatom(s), is an element otherthan carbon. Heteroatoms are typically O, S or N atoms. Included withinthe scope of heterocycle, and independently selectable, are O, N, and Sheterocycle ring structures. The ring structure may include compoundshaving one or more ring structures, such as mono-, bi-, or tricycliccompounds. In some embodiments, the heterocycle groups may be selectedfrom heterocycle groups that contain one or more heteroatoms, two ormore heteroatoms, three or more heteroatoms, or four or moreheteroatoms. Example of heterocycle groups include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl or tetrahydrothiopyranyl and the like. In certainembodiments, the heterocycle may optionally be substituted.

As used herein, “alkanoyl” generally refers to a group with thestructure —C(O)—R. In certain embodiments, R may be a hydrogen, analkyl, an 4-morpholinyl group, or a thiazoleamino group.

As used herein, “alkoxy” generally refers to a group with the structure—O—R. In certain embodiments, R may be an alkyl group, such as a C₁ toC₅ alkyl group.

For the purposes of this invention, halo substituents may beindependently selected from the halogens such as fluorine, chlorine,bromine, iodine, and astatine.

In certain preferred embodiments, X may be hydrogen, methoxy, hydroxyl,benzoxy, or a halogen, preferably bromide or chloride. In otherembodiments, X may preferably be a C₁ to C₄ alkyl or a haloalkyl.

R₁ may preferably be a C₆ to C₈ aryl group, optionally substituted withat least one R₀ group. R₀ may then preferably be methoxy, benzoxy, a C₁to C₆ alkyl, a 5 to 6 membered heteroaryl (such as furyl or imidazole),cyano, nitro, tri-fluro methyl, or a halogen, more preferably methoxy,benzoxy, iso-butyl or a halogen, and more preferably methoxy, iso-butyl,bromide or chloride. Alternatively, R₁ may be a 5 to 10 memberedheteroaryl or 3 to 12 membered heterocycle, such as a pyridinyl group, athiophene group, a furyl group, a tetrahydro furyl group, and a thiazolegroup dihydro-chromen-4-onyl group, a 1H-isoindolyl group, or abenzodioxole group.

R₂ may preferably be a —CH₂-furyl group, a pyrimidyl group, or a—C(O)O—R_(d) group. R_(d) may preferably then be a C₁ to C₆ alkyl,optionally substituted with at least one halogen; or a C₅ to C₆ aryl,optionally substituted with at least one methyl, methoxy, or halogen.

Preferred R₁ substituents also include the following, where the *indicates the bond of attachment to the carboline scaffold molecule.

Other preferred R₁ substituents include the following, where the *indicates the bond of attachment to the carboline scaffold molecule.

Preferred R₂ substituents also include the following, where the *indicates the bond of attachment to the carboline scaffold molecule.

Other preferred R₂ substituents include the following, where the *indicates the bond of attachment to the carboline scaffold molecule.

Preferred R₃ substituents include the following, where the * indicatesthe bond of attachment to the carboline scaffold molecule.

A preferred class of compounds within Formula (I) include thosecompounds of Formula (I-a) as shown below.

wherein X, R₁ and R₂ are defined as described with regard to Formula (I)and the preferred embodiments described above.

Another preferred class of compounds within Formula (I) include thosecompounds of Formula (I-b) as shown below.

wherein:

X is a halogen;

R₂ is as described above with regard to Formula (I);

R₀ is as described above with regard to Formula (I);

m is 0, 1, 2, or 3; and

n is 0, 1, 2, or 3.

Other preferred classes of compounds within Formula (I) include thefollowing.

It is understood that substituents X and R₁, R_(c), R_(d), and R_(e) ofthe compounds of Formulas (I-c) to (I-i) are defined as in Formula (I).

In other embodiments, preferred compounds of the present inventionuseful in the inhibition of VEGF production include those of Formulas(I-i) through (I-1), as shown below. In the embodiments of Formulas(I-j)through (I-1), substituents X, R₁, R₂, R₃, etc. are defined as inFormula (I), as well as Formulas (I-a) to (I-i).

Also included within the scope of the invention are pharmaceuticallyacceptable salts, hydrates, solvates, calthrates, polymorphs, racematesand stereoisomers of the compounds described herein.

In another aspect of the invention, preferred compounds of the presentinvention useful in the inhibition of VEGF production include those ofFormula (I-1) as shown below.

wherein,

X is hydrogen; a hydroxyl group; a halogen; a C₁-C₄ alkyl; a C₁ to C₅alkoxy, optionally substituted with a C₆ to C₈ aryl group;

R₁ is a hydroxyl group; a C₁ to C₈ alkyl group, optionally substitutedwith a C₆ to C₈ aryl group, wherein the C₆ to C₈ aryl group isoptionally substituted with at least one R₀ group; a heterocycle group;a heteroaryl group; and a C₆ to C₈ aryl group, optionally substitutedwith at least one R₀ group;

R_(D) is a halogen; a C₁ to C₆ alkyl, optionally substituted with one ormore halogen groups; a cyano group; a nitro group; an amino group; anaminoalkyl group; an acetamide group; an imidazole group; or OR_(a);

R_(a) is hydrogen; a C₁ to C₆ alkyl, optionally substituted with aheterocycle group or a C₆ to C₈ aryl group; or a —C(O)O—R_(b);

R_(b) is C₁ to C₄ alkyl group;

R₂ is a hydrogen; a hydroxyl; a heteroaryl group; a C₁ to C₈ alkylgroup, optionally substituted with an alkoxy, hydroxyl, heteroaryl, orC₆ to C₈ aryl group; a —C(O)—R_(e) group; a —C(O)O—R_(d) group; a—C(O)NH—R_(d) group; a —C(S)NH—R_(d) group; a —S(O₂)—R_(e) group; or(1S)-isopropyl-carbamic acid tert-butyl ester;

R_(e) is hydrogen; a 4-morpholinyl group; a thiazoleamino group; apiperazinyl group, optionally substituted with a —C(O)CH₃ group; a C₁ toC₆ alkyl group, optionally substituted with a halogen, an alkoxy, orhydroxyl group;

R_(d) is hydrogen; a benzyl group; a C₁ to C₈ alkyl group, optionallysubstituted with a halogen or an alkoxy group; a C₆ to C₈ aryl group,optionally substituted with at least one halogen, C₁ to C₅ alkyl,—C(O)OR_(e), or OR_(e);

R_(e) is a hydrogen; a C₁ to C₆ alkyl group, optionally substituted withat least one halogen or alkoxy group; or a C₆ to C₈ aryl group; and

n is 0, 1, 2, or 3.

In another embodiment, compounds of Formulas (II), (III) and (IV) areprovided, which are useful for inhibiting VEGF production, and treatingcancer.

Wherein X, R₁, R₂, R_(o) and R_(d) are defined as described above withregard with Formula (I).

For the purposes of this invention, where one or more functionalitiesencompassing X R₁, R₂, R₀, R_(a), R_(b), R_(c), R_(d), and R_(e), areincorporated into a molecule of Formulas (I), (II), and (III), includingFormulas (I-a) to (I-k), each of the functionalities appearing at anylocation within the disclosed may be independently selected, and asappropriate, independently substituted. Further, where a more genericsubstituent is set forth for any position in the molecules of thepresent invention, it is understood that the generic substituent may bereplaced with more specific substituents, and the resulting moleculesare within the scope of the molecules of the present invention.

Preferred compounds of the invention include the following.

In certain embodiments, preferred compounds include those with an EC₅₀in the VEGF ELISA assay described in Example 2 of less than about 2 uM,more preferably between about 2 uM and about 0.04 uM (200 nM to 40 nM);more preferably from about 0.04 uM to about 0.008 uM to (40 nM to 8 nM);and more preferably less than about 0.008 uM (<8 nM). Particularlypreferred compounds are Compound Nos: 2, 4, 5, 7, 8, 10, 11, 12, 17, 23,25, 81, 102, 112, 140, 328, 329, 330, 331, 332, 355, 816, 817, 818, 823,824, 825, 830, 831, 832, 837, 838, 841, 842, 843, and regioisomersthereof. In one embodiment, the preferred compounds of the inventionform a racemic mixture, and in another embodiment the compounds of theinvention are the (R), (S), (R,R), (S,S), (R,S), (S,R) isomer, in anenantiomerically pure composition. More preferably, the compounds of theinvention are the (S) isomers, in an enantiomerically pure composition.

The above compounds are listed only to provide examples that may be usedin the methods of the invention. Based upon the instant disclosure, theskilled artisan would recognize other compounds intended to be includedwithin the scope of the presently claimed invention that would be usefulin the methods recited herein.

B. Preparation of Compounds of the Invention

Compounds of the invention may be produced in any manner known in theart. By way of example, compounds of the invention may be preparedaccording to the following general schemes. More specifically, Scheme Imay be used to make compounds of Formula I. Scheme Ia can be used whenin conjunction with Scheme I when R₂ is a —CH₂-furanyl group.Alternatively, for asymmetric synthesis when R₂ is hydrogen or hydroxyl, Scheme Ib may be used.

Scheme II can be used to prepare compounds of Formula I-h.

Schemes IIIa or IIIb can be used to prepare compounds of Formula I-i.

Ref: Chem. Pharm. Bull. 1987, 4700.

Ref: Magid Abou-Gharbia et al, J. Med. Chem. 1987, 30, 1818.

In a preferred embodiment, compounds of the invention may be resolved toenantiomerically pure compositions using any method known in art. By wayof example, compounds of the invention may be resolved by directcrystallization of enantiomer mixtures, by diastereomer salt formationof enantiomers, by the formation of diasteriomers and separation, or byenzymatic resolution.

In a preferred embodiment, compounds of the invention may be resolvedthrough crystallization using, e.g., N-acetyl-L-phenylalanine to obtainthe (S) isomer, or N-acetyl-D-phenylalanine to obtain the (R) isomer, ina manner similar to that illustrated in Scheme IV.

In certain embodiments, exemplary methods of Scheme I for preparingpreferred compounds of Formula I involve the formation of free aminePictet-Spengler reaction products/intermediates, as described below inProcedure-I.

In one embodiment, Procedure-I may involve adding a desired Aldehyde(II) to a suspension of 5-substituted tryptamine. HCl (I) in 0.1Nsulfuric acid. The solution may then be stirred at about 110° C.-120° C.in a closed reaction vessel until the reaction is sufficient tocomplete, e.g., for about 15 minutes to about 20 hours. After completionof the reaction, the reaction mixture may be cooled to room temperatureand the precipitated salt may be filtered. The filtered residue may thenbe washed with ether, EtOAc or a mixture of DCM and DMF and dried togive the product (III) as acid salt. Alternatively, a desired Aldehyde(II) may be added to a suspension of 5-substituted tryptamine.HCl (I) inacetic acid and refluxed until the reaction is sufficiently complete,e.g., for about 15 minutes to about 20 hours. After completion of thereaction, the reaction mixture may be cooled to room temperature and theacid salt may be filtered. The filtered residue may then be washed withacetic acid followed by DCM and dried to give the product (III) as acidsalt. The free amine(III) may be obtained by extraction with EtOAc andwashing with aqueous ammonium hydroxide or 1M aq. sodium hydroxide.

The free amine, or its salt, may then be used to form other preferredcompounds of Formula I, such as carbamate analogs (Formula I-c,Procedure-II), amide analogs, including N-acetyl analogs (Formula 1-c,Procedure-IIIa and Procedure-IIIb), urea and thiourea analogs (FormulaI-e and I-f, Procedure-IV and Procedure-V respectively), sulfoxideanalogs (Formula 1-g, Procedure-VI), and pyrimidine analogs(Procedure-VII).

More particularly, Procedure-II may be used to synthesize carbamateanalogs of free amines(III), or their salts.

In accordance with Procedure-II, diisopropylethylamine (DIEA) may beadded to the free amine(III), or its acid salt in dichloromethane (DCM),followed by slow addition of substituted chloroformate. The reactionmixture may be stirred at room temperature for about 1 to 20 hours. Thesolvent may then be evaporated and the crude product may either bepurified by HPLC or silica gel column chromatography.

Procedure-Ma may be used to synthesize amide analogs of free amine(III),or their salts.

In accordance with Procedure-IIIa, a 15 min pre-stirred mixture of anR₂-acid and diisopropyl carbodiimide (DIC) may be added to the freeamine(III), or its acid salt in DCM and DIEA. The reaction mixture maybe stirring for about 1 h. The solvents may then be evaporated and thecrude product purified by HPLC.

Alternatively, Procedure-IIIb may be used to synthesize N-acetyl analogsof free amines (III), or their salts.

In accordance with Procedure-IIIb, pyridine may be added to the freeamine(III), or its acid salt in DCM, followed by acetic anhydride. Thereaction mixture may be stirred at room temperature for about 8 to 20hours. The solvents may then be evaporated and the crude product waspurified by HPLC.

Procedure-IV may be used to synthesize urea analogs of free amines(III),or their salts.

In accordance with Procedure-IV, DIEA and R₂-isocyanate may be added tothe free amine(III), or its acid salt in DCM. The reaction mixture maybe refluxed for about 1.5 h. The solvents may then be evaporated and thecrude product purified by HPLC.

Procedure-V may be used to synthesize thiourea analogs of freeamines(III), or their salts.

In accordance with Procedure-V, DIEA and R₂-isothiocyanate may be addedto the free amine(III), or its acid salt in DCM. The reaction mixturemay be refluxed for about 12 h. The solvents may then be evaporated andthe crude product purified by HPLC.

Procedure-VI may be used to synthesize sulfonyl analogs of freeamines(III), or their salts.

In accordance with Procedure-VI, DIEA and R₂-sulfonylchloride may beadded to the free amine(III), or its acid salt in DCM. The reactionmixture may be stirred at room temperature for about 12 h. The solventsmay then be evaporated and the crude product purified by HPLC.

Procedure-VII may be used to synthesize pyrimidine analogs of freeamines(III), or their salts.

In accordance with Procedure-VII, triethylamine and 2-bromopyrimidine inN,N-dimethylformamide (DMF) may be added to the free amine(III), or itsacid salt in DCM. The reaction mixture may be heated to about 120° C.for about 12 h. The solvents may then be evaporated and the crudeproduct purified by HPLC.

These and other reaction methodologies may be useful in preparing thecompounds of the invention, as recognized by one of skill in the art.Various modifications to the above schemes and procedures will beapparent to one of skill in the art, and the invention is not limitedspecifically by the method of preparing the compounds of the invention.

C. Methods of the Invention

In another aspect of the invention, methods are provided for theinhibition of VEGF production, the inhibition of angiogenesis, and/orthe treatment of cancer, diabetic retinopathy, rheumatoid arthritis,psoriasis, atherosclerosis, chronic inflammation, other chronicinflammation-related diseases and disorders, obesity, or exudativemacular degeneration using the compounds described herein.

In one embodiment, the invention is directed to methods for inhibitingVEGF production comprising administering a VEGF-expression inhibitingamount of at least one compound of the invention to a subject in needthereof.

In another embodiment, methods for inhibiting angiogenesis are providedcomprising administering an anti-angiogenic amount of at least onecompound of the invention to a subject in need thereof.

In yet another embodiment, methods for treating cancer, diabeticretinopathy, rheumatoid arthritis, psoriasis, atherosclerosis, chronicinflammation, other chronic inflammation-related diseases and disorders,obesity, or exudative macular degeneration are provided comprisingadministering a therapeutically effective amount of at least onecompound of the invention to a subject in need thereof.

In yet a further embodiment, the cancers which can be treated byadministering a therapeutically effective amount of at least onecompound of the invention to a subject in need thereof include solidtumor cancers. Solid tumor cancers that can be treated by the presentinvention include solid tumor carcinomas and solid tumor sarcomas. Solidtumor carcinomas include, but are not limited to, pediatric solidtumors, such as Wilms tumor and neuroblastoma, and carcinomas of theepidermis, such as malignant melanomas, as well as lung cancers,cervical cancers, colon cancers and renal cancers. Solid tumor sarcomasinclude, but are not limited to, fibrosarcomas. The methods of treatingcancer can further include the administration of one or more additionalcancer agents.

In yet another embodiment of the invention, methods for treating a solidtumor cancer by slowing tumorigenesis at a pre-vascular stage areprovided, comprising administering a therapeutically effective amount ofat least one compound of the invention to a subject in need thereof,either alone or together with one or more additional cancer agents.

In another embodiment of the invention, methods for treating a solidtumor cancer by blocking VEGF mRNA translation are provided, comprisingadministering a therapeutically effective amount of at least onecompound of the invention to a subject in need thereof, either alone ortogether with one or more additional cancer agents.

In yet another embodiment of the invention methods for treating a solidtumor cancer by reducing tumor VEGF levels are provided, comprisingadministering a therapeutically effective amount of at least onecompound of the invention to a subject in need thereof, either alone ortogether with one or more additional cancer agents.

In yet a further embodiment of the invention, methods for treating asolid tumor cancer by reducing perivascularly sequestered VEGF areprovided, comprising administering a therapeutically effective amount ofat least one compound of the invention to a subject in need thereof,either alone or together with one or more additional cancer agents. Inthis aspect, reduced perivascularly sequestered VEGF is an in situcomparison of perivascular VEGF of tumors treated with the compound ofthe invention and tumors not treated with the compound of the invention.In a preferred aspect, reduced perivascularly sequestered VEGF iscompared with levels of perivascular VEGF in tumors treated withantibodies to VEGF.

Without intending to be limited by theory, it is believed that themethods of the present invention act through a combination of mechanismsthat modulate the activity of VEGF. In this embodiment of the invention,methods for blocking VEGF mRNA translation are provided, comprisingadministering a therapeutically effective amount of at least onecompound of the invention to a subject in need thereof.

In another embodiment of the invention, methods for slowingtumorigenesis at a pre-vascular stage are provided, comprisingadministering a therapeutically effective amount of at least onecompound of the invention to a subject in need thereof, either alone ortogether with one or more additional cancer agents. The pre-vascularstage of tumorigenesis is clinically known as “carcinoma in situ” andtumors at this stage are characterized by their reliance on nearby bloodvessels for oxygen and diffusion of nutrients, due to the tumors absenceof its own vascular infrastructure. So, by slowing tumorigenesis at apre-vascular stage, one is preventing the development of a vascularinfrastructure in the tumor. In this embodiment of the invention,whether tumorigenesis has been slowed at the pre-vascular stage isdetermined by identifying whether the tumor has developed a vascularinfrastructure. In a preferred aspect, growth is reduced in the treatedtumors, as compared to the untreated tumors, by 90%, 80%, 70%, 60%, 50%,40%, 30%, 20% or 10%.

In yet a further embodiment of the invention, methods for reducingperivascularly sequestered VEGF are provided, comprising administering atherapeutically effective amount of at least one compound of theinvention to a subject in need thereof. In a preferred aspect,

In yet an additional embodiment of the invention, methods of diagnosingcancer by measuring tumor and/or plasma levels of VEGF are provided.Tumor levels of VEGF can be measured using biopsy tissue, where plasmaVEGF levels can be measured by taking blood. In humans, different tumorssecrete different levels of VEGF. Standard ELISA procedures can be usedto measure the amount of VEGF in the tumor or plasma. See, for example,Verheul, H. m. W. et al. (2000) Platelet and coagulation activation withvascular endothelial growth factor generation in soft tissue sacomas.Clim Cancer Res. 6:166. For tumors that do not secrete large amounts ofVEGF into the plasma, the tumor VEGF concentration can be determined todiagnose the tumor progression. For tumors that do secrete large amountof VEGF into the plasma, plasma VEGF concentration can be determined todiagnose the tumor progression. After most known cancer treatments, VEGFlevels are not affected, and therefore the plasma or tumor levels ofVEGF do not predict efficacy of the treatment (i.e., progression of thecancer). Compounds of the present invention can affect VEGF levels, bothin the plasma and tumor, and therefore measuring VEGF levels is anaccurate way to predict the progression of the cancer when the methodsof the present invention for treating cancer are used.

In yet another embodiment of the invention, methods for reducing tumoror plasma VEGF levels are provided, comprising administering atherapeutically effective amount of at least one compound of theinvention to a subject in need thereof. In this embodiment, VEGF levelscan be measured in a tumor not treated with the compounds of the presentinvention and the VEGF levels compared to the VEGF levels measured in atumor treated with the compounds of the present invention, therebyshowing that by treatment of tumors with the compounds of the presentinvention VEGF levels are reduced.

In yet another embodiment of the invention, methods for treating a solidtumor cancer are provided, comprising (a) measuring plasma VEGF levels,tumor VEGF levels, or both, and administering a therapeuticallyeffective amount of at least one compound of the invention to a subjectin need thereof. In an embodiment, a level of VEGF is measured todetermine whether treatment with a compound of the present inventionshould be undertaken. In this aspect, treatment with a compound of thepresent invention is preferred and more effective as the VEGF levelsincrease.

In yet a further embodiment of the invention, methods for treating asolid tumor cancer are provided, comprising administering atherapeutically effective amount of at least one compound of theinvention to a subject in need thereof, together with one or moreadditional cancer agents.

In preferred embodiments, the methods of the invention compriseadministering a therapeutically effective amount of at least onecompound of the invention, wherein the compound is an (5) isomer.

According to the methods of the invention, the compound(s) may beadministered to the subject via any drug delivery route known in theart. Specific exemplary administration routes include oral, ocular,rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular,intravenous (bolus and infusion), intracerebral, transdermal, andpulmonary.

The terms “VEGF-inhibiting amount”, “anti-angiogenic amount”, and“therapeutically effective amount”, as used herein, refer to an amountof a pharmaceutical agent to treat, ameliorate, or prevent theidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory affect. The affect can be detected by, for example, theassays disclosed in the following examples. The precise effective amountfor a subject will depend upon the subject's body weight, size, andhealth; the nature and extent of the condition; and the therapeutic orcombination of therapeutics selected for administration. Therapeuticallyeffective amounts for a given situation can be determined by routineexperimentation that is within the skill and judgment of the clinician.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, ED₅₀/LD₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies may be used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include an ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

More specifically, the concentration-biological effect relationshipsobserved with regard to the compound(s) of the present inventionindicate an initial target plasma concentration ranging fromapproximately 0.1 μg/mL to approximately 100 μg/mL, preferably fromapproximately 5 μg/mL to approximately 50 μg/mL, more preferably fromapproximately 5 μg/mL to approximately 10 μg/mL. To achieve such plasmaconcentrations, the compounds of the invention may be administered atdoses that vary from 0.1 μg to 100,000 mg, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and is generally available topractitioners in the art. In general the dose will be in the range ofabout 1 mg/day to about 10 g/day, or about 0.1 g to about 3 g/day, orabout 0.3 g to about 3 g/day, or about 0.5 g to about 2 g/day, insingle, divided, or continuous doses for a patient weighing betweenabout 40 to about 100 kg (which dose may be adjusted for patients aboveor below this weight range, particularly children under 40 kg).

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeagent(s) or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

D. Metabolites of the Compounds of the Invention

Also falling within the scope of the present invention are the in vivometabolic products of the compounds described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,esterification and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising contacting a compound of this inventionwith a mammalian tissue or a mammal for a period of time sufficient toyield a metabolic product thereof. Such products typically areidentified by preparing a radio-labeled (e.g. C¹⁴ or H³) compound of theinvention, administering it in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to a mammal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours), and isolating its conversion productsfrom urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS or NMR analysis. In general, analysis of metabolites may be done inthe same way as conventional drug metabolism studies well-known to thoseskilled in the art. The conversion products, so long as they are nototherwise found in vivo, are useful in diagnostic assays for therapeuticdosing of the compounds of the invention even if they possess nobiological activity of their own.

E. Pharmaceutical Compositions of the Invention

While it is possible for the compounds of the present invention to beadministered neat, it may be preferable to formulate the compounds aspharmaceutical compositions. As such, in yet another aspect of theinvention, pharmaceutical compositions useful in the methods of theinvention are provided. The pharmaceutical compositions of the inventionmay be formulated with pharmaceutically acceptable excipients such ascarriers, solvents, stabilizers, adjuvants, diluents, etc., dependingupon the particular mode of administration and dosage form. Thepharmaceutical compositions should generally be formulated to achieve aphysiologically compatible pH, and may range from a pH of about 3 to apH of about 11, preferably about pH 3 to about pH 7, depending on theformulation and route of administration. In alternative embodiments, itmay be preferred that the pH is adjusted to a range from about pH 5.0 toabout pH 8.0.

More particularly, the pharmaceutical compositions of the inventioncomprise a therapeutically or prophylactically effective amount of atleast one compound of the present invention, together with one or morepharmaceutically acceptable excipients. Optionally, the pharmaceuticalcompositions of the invention may comprise a combination of compounds ofthe present invention, or may include a second active ingredient usefulin the treatment of cancer, diabetic retinopathy, or exudative maculardegeneration.

Formulations of the present invention, e.g., for parenteral or oraladministration, are most typically solids, liquid solutions, emulsionsor suspensions, while inhaleable formulations for pulmonaryadministration are generally liquids or powders, with powderformulations being generally preferred. A preferred pharmaceuticalcomposition of the invention may also be formulated as a lyophilizedsolid that is reconstituted with a physiologically compatible solventprior to administration. Alternative pharmaceutical compositions of theinvention may be formulated as syrups, creams, ointments, tablets, andthe like.

The term “pharmaceutically acceptable excipient” refers to an excipientfor administration of a pharmaceutical agent, such as the compounds ofthe present invention. The term refers to any pharmaceutical excipientthat may be administered without undue toxicity. Pharmaceuticallyacceptable excipients are determined in part by the particularcomposition being administered, as well as by the particular method usedto administer the composition. Accordingly, there exists a wide varietyof suitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences).

Suitable excipients may be carrier molecules that include large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Other exemplary excipients includeantioxidants such as ascorbic acid; chelating agents such as EDTA;

carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethylcellulose, stearic acid; liquids such as oils, water,saline, glycerol and ethanol; wetting or emulsifying agents; pHbuffering substances; and the like. Liposomes are also included withinthe definition of pharmaceutically acceptable excipients.

The pharmaceutical compositions of the invention may be formulated inany form suitable for the intended method of administration. Whenintended for oral use for example, tablets, troches, lozenges, aqueousor oil suspensions, non-aqueous solutions, dispersible powders orgranules (including micronized particles or nanoparticles), emulsions,hard or soft capsules, syrups or elixirs may be prepared. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions, and suchcompositions may contain one or more agents including sweetening agents,flavoring agents, coloring agents and preserving agents, in order toprovide a palatable preparation.

Pharmaceutically acceptable excipients particularly suitable for use inconjunction with tablets include, for example, inert diluents, such ascelluloses, calcium or sodium carbonate, lactose, calcium or sodiumphosphate; disintegrating agents, such as croscarmellose sodium,cross-linked povidone, maize starch, or alginic acid; binding agents,such as povidone, starch, gelatin or acacia; and lubricating agents,such as magnesium stearate, stearic acid or talc. Tablets may beuncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample celluloses, lactose, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with non-aqueousor oil medium, such as glycerin, propylene glycol, polyethylene glycol,peanut oil, liquid paraffin or olive oil.

In another embodiment, pharmaceutical compositions of the invention maybe formulated as suspensions comprising a compound of the presentinvention in admixture with at least one pharmaceutically acceptableexcipient suitable for the manufacture of a suspension. In yet anotherembodiment, pharmaceutical compositions of the invention may beformulated as dispersible powders and granules suitable for preparationof a suspension by the addition of suitable excipients.

Excipients suitable for use in connection with suspensions includesuspending agents, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methylcelluose, sodium alginate,polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or wettingagents such as a naturally occurring phosphatide (e.g., lecithin), acondensation product of an alkylene oxide with a fatty acid (e.g.,polyoxyethylene stearate), a condensation product of ethylene oxide witha long chain aliphatic alcohol (e.g., heptadecaethyleneoxycethanol), acondensation product of ethylene oxide with a partial ester derived froma fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitanmonooleate); and thickening agents, such as carbomer, beeswax, hardparaffin or cetyl alcohol. The suspensions may also contain one or morepreservatives such as acetic acid, methyl and/or n-propylp-hydroxy-benzoate; one or more coloring agents; one or more flavoringagents; and one or more sweetening agents such as sucrose or saccharin.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth;naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids; hexitol anhydrides, such assorbitan monooleate; and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents. Syrups andelixirs may be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such formulations may also contain a demulcent, apreservative, a flavoring or a coloring agent.

Additionally, the pharmaceutical compositions of the invention may be inthe form of a sterile injectable preparation, such as a sterileinjectable aqueous emulsion or oleaginous suspension. This emulsion orsuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, such as a solution in 1,2-propane-diol.The sterile injectable preparation may also be prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid may likewise be used in the preparation of injectables.

Generally, the compounds of the present invention useful in the methodsof the present invention are substantially insoluble in water and aresparingly soluble in most pharmaceutically acceptable protic solventsand in vegetable oils. However, the compounds are generally soluble inmedium chain fatty acids (e.g., caprylic and capric acids) ortriglycerides and have high solubility in propylene glycol esters ofmedium chain fatty acids. Also contemplated in the invention arecompounds which have been modified by substitutions or additions ofchemical or biochemical moieties which make them more suitable fordelivery (e.g., increase solubility, bioactivity, palatability, decreaseadverse reactions, etc.), for example by esterification, glycosylation,PEGylation, etc.

In a preferred embodiment, the compounds of the present invention may beformulated for oral administration in a lipid-based formulation suitablefor low solubility compounds. Lipid-based formulations can generallyenhance the oral bioavailability of such compounds. As such, a preferredpharmaceutical composition of the invention comprises a therapeuticallyor prophylactically effective amount of a compound of the presentinvention, together with at least one pharmaceutically acceptableexcipient selected from the group consisting of: medium chain fattyacids or propylene glycol esters thereof (e.g., propylene glycol estersof edible fatty acids such as caprylic and capric fatty acids) andpharmaceutically acceptable surfactants such as polyoxyl 40 hydrogenatedcastor oil.

In an alternative preferred embodiment, cyclodextrins may be added asaqueous solubility enhancers. Preferred cyclodextrins includehydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosylderivatives of α-, β-, and γ-cyclodextrin. A particularly preferredcyclodextrin solubility enhancer is hydroxypropyl-β-cyclodextrin (HPBC),which may be added to any of the above-described compositions to furtherimprove the aqueous solubility characteristics of the compounds of thepresent invention. In one embodiment, the composition comprises 0.1% to20% hydroxypropyl-β-cyclodextrin, more preferably 1% to 15%hydroxypropyl-β-cyclodextrin, and even more preferably from 2.5% to 10%hydroxypropyl-β-cyclodextrin. The amount of solubility enhancer employedwill depend on the amount of the compound of the present invention inthe composition.

F. Combination Therapy

It is also possible to combine any compound of the present inventionwith one or more other active ingredients useful in the treatment ofcancer, including compounds, in a unitary dosage form, or in separatedosage forms intended for simultaneous or sequential administration to apatient in need of treatment. When administered sequentially, thecombination may be administered in two or more administrations. In analternative embodiment, it is possible to administer one or morecompounds of the present invention and one or more additional activeingredients by different routes.

The skilled artisan will recognize that a variety of active ingredientsmay be administered in combination with the compounds of the presentinvention that may act to augment or synergistically enhance theVEGF-inhibiting and/or anti-angiogenesis activity of the compounds ofthe invention.

More specifically, for methods involving the treatment of cancer, agentsknown in the art to be useful for treating cancer are provided. Suchagents include, but are not limited to, radiation therapy, agents thatcause DNA damage, agents that reduce the concentration or effect of agrowth factor, agents that inhibit angiogenesis, paclitaxel,fluorouracil, CPT-11, a tyrosine kinase inhibitor, a COX-2 inhibitor,thalidomide, gemcitabine, squalamine, endostatin, angiostatin, AE-941,lenalidomide, medi-522, 2-methoxyestradiol, carboxyamidotriazole,combretastatin A4 phosphate, SU6668, SU11248, BMS-275291, COL-3,cilengitide, IMC-1121B, vatalanib, LY317615, VEGF Trap, ZD6474,halofuginone, hydrobromide, celecoxib, interferon alpha, interleukin-12,and antibodies capable of binding VEGF or a VEGF receptor, such asbevacizumab. VEGF receptors include VEGF receptor 1, VEGF receptor 2,and VEGF receptor 3. In another embodiment, the compounds of the presentinvention are used in combination with an agent that blocks the activityof a VEGF receptor. In yet another embodiment, the compounds of thepresent invention can be used in combination with agents that can blockthe VEGF signaling pathway. Treatment only with a factor that can blockVEGF signaling may cause an increase in VEGF concentration. In such acase, including a compound of the present invention in the treatmentprotocol can prevent the subsequent increase in VEGF levels. Similarly,use of the compounds of the present invention in combination with anantibody is highly preferred. Antibodies are relatively large and maynot cross tight barriers, allowing secreted VEGF to remain in areas suchas the perivascular space. Post-transcriptional control of VEGFexpression can prevent the tumor from retaining as much VEGF in theperivascular space, in the extracellular matrix, or in other spaces andvessels that have a physical barrier to antibodies.

According to the methods of the invention, the combination of activeingredients may be: (1) co-formulated and administered or deliveredsimultaneously in a combined formulation; (2) delivered by alternationor in parallel as separate formulations; or (3) by any other combinationtherapy regimen known in the art. When delivered in alternation therapy,the methods of the invention may comprise administering or deliveringthe active ingredients sequentially, e.g., in separate solution,emulsion, suspension, tablets, pills or capsules, or by differentinjections in separate syringes. In general, during alternation therapy,an effective dosage of each active ingredient is administeredsequentially, i.e., serially, whereas in simultaneous therapy, effectivedosages of two or more active ingredients are administered together.Various sequences of intermittent combination therapy may also be used.

To assist in understanding the present invention, the following Examplesare included. The experiments relating to this invention should not, ofcourse, be construed as specifically limiting the invention and suchvariations of the invention, now known or later developed, which wouldbe within the purview of one skilled in the art are considered to fallwithin the scope of the invention as described herein and hereinafterclaimed.

Examples

The present invention is described in more detail with reference to thefollowing non-limiting examples, which are offered to more fullyillustrate the invention, but are not to be construed as limiting thescope thereof. The examples illustrate the preparation of certaincompounds of the invention, and the testing of these compounds in vitroand/or in vivo. Those of skill in the art will understand that thetechniques described in these examples represent techniques described bythe inventors to function well in the practice of the invention, and assuch constitute preferred modes for the practice thereof. However, itshould be appreciated that those of skill in the art should in light ofthe present disclosure, appreciate that many changes can be made in thespecific methods that are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Preparation of Compounds of the Invention

Using the schemes and procedures described above in Section B, one mayprepare certain compounds of the invention as follows. Other preferredcompounds of the invention, such as those in Table 5 below, may besimilarly prepared.

Example 1A Compounds of Formula I, Scheme I

Certain compounds of Formula I may be prepared according to Scheme Iusing free amine products/intermediates, or their salts prepared inaccordance with Procedure I. By way of example, certain freeamines(III), or their salts are prepared using Procedure I. Table 4illustrates certain free amines(III) or their salts, Intermediates 1-11.

TABLE 4 (III)

R- R₁- of Free of Free Intermediate Amine (III) Amine (III) 1 Cl 4-OMe 2Cl 2,3-difluoro 3 Cl 4-Cl 4 Cl 4-CN 5 Cl 4-F 6 Cl 4-iPr 7 Br 4-Cl 8 Br4-Me 9 Br 4-iPr 10 Br 3-Cl 11 Br 4-OMe 12 Cl 4-(2-morpholine-4-yl-ethoxy)

Intermediate-1:

This intermediate is prepared using Procedure-I with5-chlorotryptamine.HCl (5.8 g, 25 mmol), p-anisaldehyde (6.13 mL, 50mmol) and 0.1N sulfuric acid (60 mL) to give the title compound as anacid salt (6.1 g, 59%). ES-MS: 313 (M+H)⁺. Alternatively, thisintermediate is prepared using Procedure-1B with 5-chlorotryptamine.HCl(20 g, 86.5 mmol), p-anisaldehyde (15.9 mL, 130 mmol) and acetic acid(250 mL) to give the title compound as an acid salt (25.8 g, 79%).ES-MS: 313 (M+H)⁺.

Intermediate-2:

This intermediate is prepared using Procedure-I with5-chlorotryptamine.HCl (116 mg, 0.5 mmol), 2,3-difluoro benzaldehyde(109 μL, 1 mmol) and 0.1N sulfuric acid (2 mL) to give the titlecompound as an acid salt (158 mg, 75%). ES-MS: 319 (M+H)⁺

Intermediate-3:

This intermediate is prepared using Procedure-I with5-chlorotryptamine.HCl (462 mg, 2 mmol), 4-chloro benzaldehyde (562 mg,4 mmol) and 0.1N sulfuric acid (8 mL) to give the title compound as anacid salt (825 mg, 99%). ES-MS: 317 (M+H)⁺

Intermediate-4:

This intermediate is prepared using Procedure-I with5-chlorotryptamine.HCl (462 mg, 2 mmol), 4-cyano benzaldehyde (525 mg, 4mmol) and 0.1N sulfuric acid (8 mL) to give the title compound as anacid salt (810 mg, 100%). ES-MS: 308 (M+H)⁺

Intermediate-5:

This intermediate is prepared using Procedure-I with5-chlorotryptamine.HCl (374 mg, 1.5 mmol), 4-fluoro benzaldehyde (322μL, 3 mmol) and 0.1N sulfuric acid (4 mL) to give the title compound asan acid salt (250 mg, 42%). ES-MS: 301 (M+H)⁺

Intermediate-6:

This intermediate is prepared using Procedure-I with5-chlorotryptamine.HCl (1.15 g, 5 mmol), 4-isopropyl benzaldehyde (1.516mL, 10 mmol) and 0.1N sulfuric acid (12 mL) to give the title compoundas an acid salt (628 mg, 30%). ES-MS: 325 (M+H)⁺

Intermediate-7:

This intermediate is prepared using Procedure-I with5-bromotryptamine.HCl (551 mg, 2 mmol), 4-chloro benzaldehyde (562 mg, 4mmol) and 0.1N sulfuric acid (8 mL) to give the title compound as anacid salt (330 mg, 36%). ES-MS: 363 (M+H)⁺

Intermediate-8:

This intermediate is prepared using Procedure-I with5-bromotryptamine.HCl (551 mg, 2 mmol), p-tolualdehyde (471 μL, 4 mmol)and 0.1N sulfuric acid (8 mL) to give the title compound as hydrogensulfate salt (257 mg, 29%). ES-MS: 341 (M+H)⁺. Alternatively, thisintermediate is prepared using Procedure-1B with 5-bromotryptamine.HCl(10 g, 36.3 mmol), p-tolualdehyde (6.41 mL, 54.5 mmol) and acetic acid(120 mL) to give the title compound as acetate salt (14.5 g, 100%).ES-MS: 341 (M+H)⁺

Intermediate-9 (Compound 112)

This product/intermediate is prepared using Procedure-I with5-bromotryptamine.HCl (551 mg, 2 mmol), 4-isopropyl benzaldehyde (606μL, 4 mmol) and 0.1N sulfuric acid (8 mL) to give the title compound ashydrogen sulfate salt (329 mg, 35%). ES-MS: 369 (M+H)⁺. Alternatively,this intermediate is prepared using Procedure-1B with5-bromotryptamine.HCl (10 g, 36.3 mmol), 4-isopropyl benzaldehyde (8.24mL, 54.5 mmol) and acetic acid (120 mL) to give the title compound asacetate salt (13 g, 77%). ES-MS: 369 (M+H)⁺

Intermediate-10:

This intermediate is prepared using Procedure-I with5-bromotryptamine.HCl (551 mg, 2 mmol), 3-chloro benzaldehyde (453 μL, 4mmol) and 0.1N sulfuric acid (8 mL) to give the title compound as anacid salt (662 mg, 72%). ES-MS: 361 (M+H)⁺

Intermediate-11:

This intermediate is prepared using Procedure-I with5-bromotryptamine.HCl (551 mg, 2 mmol), p-anisaldehyde (491 μL, 4 mmol)and 0.1N sulfuric acid (8 mL) to give the title compound as an acid salt(611 mg, 67%). ES-MS: 357 (M+H)⁺

Intermediate-12:

The 4-(2-Morpholin-4-yl-ethoxy)-benzaldehyde reaction intermediate isprepared by combining 4-hydroxybenzaldehyde (1.2 g, 10.0 mmol),4-(2-chloroethyl)-morpholine hydrochloride (2.0 g, 11.0 mmol), potassiumcarbonate (4.1 g, 30.0 mmol), and potassium iodide (170 mg, 1 mmol) in100 ml of acetone and heating to reflux with stirring. After all the4-hydroxybenzaldehyde is consumed (48 hours by LC/MS), the solids arefiltered and the solvent is removed in vacuo. The yield is 4.1 g.

Then Intermediate 12 is prepared in accordance with Procedure-IB. Thus,5-Chlorotryptamine hydrochloride (231 mg, 1.0 mmol) is combined with4-(2-Morpholin-4-yl-ethoxy)-benzaldehyde (565 mg, ˜1.2 mmol) in 3 mL ofglacial acetic acid. The suspension is heated to about 120° C. for 10minutes with constant cooling and a max power of 300W using the CEMExplorer microwave system. Acetonitrile (2 mL) is added to the cooledreaction mixture, and the solid is filtered and washed with 1 mL ofacetonitrile to produce the acetic acid salt of Intermediate 12(6-Chloro-1-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-2,3,4,9-tetrahydro-1H-β-carboline)(179 mg, 34%).

Intermediates 1-12 may then be used to prepare compounds of theinvention according to Procedures II through VII as follows.

Compound 2:

This product is prepared by Procedure-II using the Intermediate-1 (3 g,9.6 mmol), ethyl chloroformate (1.37 mL, 14.4 mmol) and DIEA (2.5 mL,14.4 mmol) in dichloromethane (70 mL) to give the title compound aswhite powder (1.56 g, 42%). ES-MS: 385 (M+H)⁺.

Compound 4:

This product is prepared by Procedure-II using the Intermediate-7 (72mg, 0.2 mmol), ethyl chloroformate (29 μL, 0.3 mmol) and DIEA (52 μL,0.3 mmol) in dichloromethane (2 mL) to give the title compound as whitepowder (37 mg, 43%). ES-MS: 435 (M+H)⁺.

Compound 5:

This product is prepared by the Procedure-II using the Intermediate-2(50 mg, 0.16 mmol), ethyl chloroformate (23 μL, 0.24 mmol) and DIEA (42μL, 0.24 mmol) in dichloromethane (2 mL) to give the title compound aswhite powder (25 mg, 41%). ES-MS: 391 (M+H)⁺.

Compound 7:

This product is prepared by the Procedure-II using the Intermediate-9(74 mg, 0.2 mmol), ethyl chloroformate (29 μL, 0.3 mmol) and DIEA (52μL, 0.3 mmol) in dichloromethane (2 mL) to give the title compound aswhite powder (34 mg, 38%). ES-MS: 441 (M+H)⁺.

Compound 8:

This product is prepared by the Procedure-II using the Intermediate-8(72 mg, 0.2 mmol), ethyl chloroformate (29 μL, 0.3 mmol) and DIEA (52μL, 0.3 mmol) in dichloromethane (2 mL) to give the title compound aswhite powder (39 mg, 47%). ES-MS: 413 (M+H)⁺.

Compound 10:

This product is prepared by the Procedure-II using the Intermediate-1acetate (10.5 g, 28.2 mmol), 4-chlorophenyl chloroformate (4.74 mL, 33.8mmol) and DIEA (9.8 mL, 56.4 mmol) in dichloromethane (300 mL) to givethe title compound as white powder (10.2 g, 78%). ES-MS: 467 (M+H)⁺.

Compound 11:

This product is prepared by the Procedure-II using the Intermediate-3(63 mg, 0.2 mmol), ethyl chloroformate (29 μL, 0.3 mmol) and DIEA (52μL, 0.3 mmol) in dichloromethane (2 mL) to give the title compound aswhite powder (31 mg, 40%). ES-MS: 389 (M+H)⁺.

Compound 12:

This product is prepared by the Procedure-II using the Intermediate-4(31 mg, 0.1 mmol), 2-chloroethyl chloroformate (16 μL, 0.15 mmol) andDIEA (26 μL, 0.15 mmol) in dichloromethane (2 mL) to give the titlecompound as white powder (22 mg, 53%). ES-MS: 414 (M+H)⁺.

Compound 17:

This product is prepared by the Procedure-II using the Intermediate-1(47 mg, 0.15 mmol), 4-methylphenyl chloroformate (33 μL, 0.23 mmol) andDIEA (39 μL, 0.23 mmol) in dichloromethane (2 mL) to give the titlecompound as white powder (34 mg, 51%). ES-MS: 447 (M+H)⁺.

Compound 23:

This product is prepared by the Procedure-II using the Intermediate-5(30 mg, 0.1 mmol), ethyl chloroformate (14 μL, 0.15 mmol) and DIEA (26μL, 0.15 mmol) in dichloromethane (2 mL) to give the title compound aswhite powder (21 mg, 56%). ES-MS: 373 (M+H)⁺.

Compound 25:

This product is prepared by the Procedure-VII using the Intermediate-9(74 mg, 0.2 mmol), 2-bromopyrimidine (48 mg, 0.3 mmol) andtriethylamine(42 μL, 0.3 mmol) in DMF (2 mL) to give the title compound(42 mg, 47%). ES-MS: 447 (M+H)⁺.

Compound 102:

This product is prepared by the Procedure-IIIb using the Intermediate-9(74 mg, 0.2 mmol), acetic anhydride (47 μL, 0.5 mmol) and pyridine (41μL, 0.5 mmol) in dichloromethane (2 mL) to give the title compound aswhite powder (31 mg, 38%). ES-MS: 411 (M+H)⁺.

Compound 140:

This product is prepared by the Procedure-IV using the Intermediate-10(72 mg, 0.2 mmol), cyclohexyl isocyanate (26 μL, 0.2 mmol) and DIEA (37μL, 0.21 mmol) in dichloromethane (2 mL) to give the title compound aswhite powder (51 mg, 53%). ES-MS: 486 (M+H)⁺.

Compound 166:

This product is prepared by the Procedure-Ma using its free amineintermediate (141 mg, 0.5 mmol), Boc-L-Alanine (105 mg, 0.6 mmol), DIC(94 μL, 0.6 mmol), DIEA (105 μL, 0.6 mmol) and dichloromethane (4 mL) togive the title compound (105 mg, 46%). ES-MS: 420 (M+H)⁺.

Compound 225:

This product is prepared by the Procedure-VI using its free amineintermediate (78 mg, 0.2 mmol), methyl sulfonylchloride (16 μL, 0.2mmol) and DIEA (37 μL, 0.21 mmol) and dichloromethane (2 mL) to give thetitle compound (32 mg, 34%). ES-MS: 461 (M+H)⁺.

Compound 242:

This product is prepared by the Procedure-V using its free amineintermediate (59 mg, 0.2 mmol), cyclohexyl isothiocyanate (29 μL, 0.2mmol), DIEA (35 μL, 0.2 mmol) and dichloromethane (4 mL) to give thetitle compound (52 mg, 60%). ES-MS: 438 (M+H)⁺.

Compound 279:

This product is prepared by generating Intermediate 12(6-Chloro-144-(2-morpholin-4-yl-ethoxy)-phenyl]-2,3,4,9-tetrahydro-1H-β-carboline)using Procedure-I. Intermediate 12 is then used to generate Compound 279(6-Chloro-1-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-1,3,4,9-tetrahydro-b-carboline-2-carboxylicacid ethyl ester) using Procedure-II.

In accordance with Procedure-II, Intermediate 12 (82 mg, 0.20 mmol),ethyl chloroformate (24 mg, 21 μL, 0.22 mmol), anddiisopropylethylamine(175 μL, 1.00 mmol) are dissolved in methylenechloride (2 mL) and stirred at room temperature for 15 minutes to formCompound 279. The solvent is removed under a stream of nitrogen. Thecrude mixture is purified by preparative reversed phase HPLC on a C-18column using a gradient of acetonitrile in water buffered with 0.2%trifluoroacetic acid (TFA). The TFA salt of Compound 279 (3.7 mg, 3%) isisolated as a yellow solid. The same procedure may be applied for othercarbamate formation reactions according to Procedure-II.

Compound 320:

This product/intermediate is prepared using Procedure-I with 5-benzyloxytryptamine.HCl (100 mg, 0.33 mmol), pyridine-3-carboxaldehyde (62 μL,0.66 mmol) and 0.1N sulfuric acid (2 mL) to give the title compound asdihydrogen sulfate salt (64 mg, 55%). ES-MS: 356 (M+H)⁺

Compound 329:

This product is prepared by the Procedure-VII using the Intermediate-11(71 mg, 0.2 mmol), 2-bromopyrimidine (48 mg, 0.3 mmol) andtriethylamine(42 μL, 0.3 mmol) in DMF (2 mL) to give the title compound(41 mg, 49%). ES-MS: 434 (M+H)⁺.

Compound 330:

This product is prepared by the Procedure-II using the Intermediate-6(65 mg, 0.2 mmol), 2-fluoroethyl chloroformate (38 μL, 0.3 mmol) andDIEA (70 μL, 0.4 mmol) in dichloromethane (2 mL) to give the titlecompound as white powder (34 mg, 41%). ES-MS: 415 (M+H)⁺.

Compound 332:

This product is prepared by the Procedure-II using the Intermediate-7(36 mg, 0.1 mmol), 4-methoxyphenyl chloroformate (22 μL, 0.15 mmol) andDIEA (26 μL, 0.15 mmol) in dichloromethane (2 mL) to give the titlecompound as white powder (41 mg, 81%). ES-MS: 511 (M+H)⁺.

Example 1B Certain Starting Materials, Scheme Ia

Scheme Ia can be used when in conjunction with Scheme I (above) togenerate starting materials when R₂ is a —CH₂-furanyl group, as follows.

2-furaldehyde (0.05 mL, 1.1 eq) is added to a solution of5-chlorotryptamine(114 mg, 0.586 mmol) in 2 mL of MeOH. The reactionmixture is stirred at room temperature for about 1 hour. NaBH₄ (110 mg,5 eq) is added slowly. The reaction mixture is stirred at roomtemperature for about 30 min. MeOH is evaporated and the residue ispartitioned between water and methylene chloride. The organic layer isseparated and dried over K₂CO₃. The collected organic layer isconcentrated to give 134.9 mg of viscous oil (84%).

Example 1C Compounds of Formula I, Scheme Ib

Alternatively, certain compounds of Formula I may be prepared accordingto Scheme Ib as follows.

A suspension of reaction material A (8.05 g, 35.9 mmol) and CH₃COONH₄(4.15 g, 1.5 eq) in 60 mL of CH₃NO₂ is refluxed in oil bath at about110° C. After about 30 minutes, the reaction mixture is cooled withice-bath. The precipitated solid is filtered and washed with water(3×100 mL), followed by hexane (2×50 mL) to give crude indole product B.The collected solid is dried under vacuum at about 40° C. for about 30min to give 6.97 g of brown solid (73%).

A solution of indole product B (12.32 g, 46.1 mmol) in THF (130 mL) isthen treated with a solution of tetrabutylammonium borohydride (11.9 g,1 eq) in 75 mL of THF slowly for about 60 minutes at about −5° C. Thereaction is stirred at room temperature for about 1 hour and dilutedwith dichloromethane (200 mL). The organic layer is washed with watertwice and brine. The combined organic layers are dried and evaporatedunder vacuum. The residue is purified on silica gel to give 10.28 g ofsolid C (83%).

Ammonium chloride (9.9 mL of aqueous solution (100 mg/mL), 2 eq) and Zn(725 mg, 1.2 eq) are then added to a solution of indole product C (2.49g, 9.24 mmol) in 161 mL of THF. The reaction mixture is stirred at roomtemperature for about 10 min and Zn (725 mg, 1.2 eq) is then added.After about 30 min, additional Zn (967 mg, 1.6 eq) is added and stirredfor about 2 hours, followed by the addition of further Zn (845 mg, 1.4eq). After stirring at room temperature for about 15 min, Zn is filteredoff and the residue is concentrated and dissolved in THF. The resultingsolution is then treated with p-chlorobenzaldehyde (0.7 eq) and stirredat room temperature for about 15 hours. The reaction mixture isconcentrated under vacuum and purified on silica gel to give 953.5 mg ofthe desired nitrone product D.

(+)-DIP-Cl (6.93 mL, 2 eq, 85.8 mg/mL in CH₂Cl₂) is then added to asolution of nitrone product D (350 mg, 0.93 mmol) in 60 mL ofdichloromethane. The reaction mixture is stirred at about −78° C. forabout 10 days and quenched with a mixture of 10% NaHCO₃ (7 mL) and 10 mLof water. The aqueous layer is extracted with dichloromethane threetimes. Combined organic layers are concentrated and purified on silicagel to give the desired hydroxylamine product E (>98% ee).

Water (11.5 mL), NH₄Cl (2.5 mL, 5 eq) and Zn (908 mg, 15 eq) are thenadded to a solution of hydroxylamine product E (0.927 mmol) in THF (28mL). The reaction mixture is stirred at room temperature for about 1day. Additional THF (10 mL), NH₄Cl (5 mL, 10 eq) and Zn (1.8 g, 30 eq)are then added and stirred for about another 21 hours. Again, THF (10mL), NH4Cl (5 mL, 10 eq) and Zn (1.8 g, 30 eq) are added and stirred forabout another 20 hours. The reaction mixture is then filtered throughcelite and washed with MC. The collected dichloromethane layer is washedwith water and brine. The organic layer is dried and concentrated togive a boron complex of beta-carboline. This product is dissolved in 20mL of THF. This solution is loaded into prepacked cation exchange resin(preconditioned with MeOH and THF) and washed with THF. The combined THFsolution is concentrated to give 390 mg of free amine. The solid is thenwashed with ether and hexane consecutively to yield 130 mg of theenantiomerically pure compound F.

Example 1D Compounds of Formula I, Scheme II

Compounds of Formula I-h may be prepared according to Scheme II asfollows.

p-anisaldehyde (2.16 g, 15.9 mmol, 1.93 mL) is added to a suspension of5-Bromotryptophan A (3 g, 10.6 mmol) in 100 mL of Acetic acid at roomtemperature. The reaction mixture is then heated to reflux at about 125° C. in silicon oil bath and maintained at that temperature for about 3hours 20 minutes. The resultant solution is concentrated under vacuum.The residue is triturated with dichloromethane, diethyl ether and hexaneto yield a powdery brown solid. The acetic salts of the intermediateproduct B is collected and washed with hexane three times.

The intermediate product B is suspended (70 mg, 0.174 mmol) in 2 mL ofdichloromethane, and triethylamine(52.8 mg, 0.522 mmol),5-methyl-2-aminothiazole (37.6 mg, 0.26 mmol) and PyBOP (135.8 mg, 0.26mmol) is added to the suspension. The reaction mixture is stirred atroom temperature for about 6 hour and quenched with sat. NaHCO₃solution. The aqueous layer is extracted with dichloromethane. Thecombined organic layers are dried over K₂CO₃ and concentrated.Purification on silica gel with 40% ethyl acetate in hexane yields 8.1mg of the desired amide C. LCMS [MH⁺] 498, Rt=2.54.

Example 1E Compounds of Formula I, Scheme III

Compounds of Formula I-i may be prepared according to Scheme III asfollows.

Tryptophan A (1.0 g, 5.0 mmol) and 3-methoxybenzaldehyde (670 μL, 5.5mmol) are suspended/dissolved in acetonitrile (100 mL) and concentratedsulfuric acid (100 μL) is added. The reaction is heated to reflux untilall the aldehyde was consumed (overnight). The solvent was removed invacuo and the residue was dissolved in 5 mL of ethanol. The product wasprecipitated out with ether, filtered, and washed with 10 mL of ether.The desired β-carboline product/intermediate B(1-(3-Methoxy-phenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylicacid) is isolated as a beige solid (1.2 g, 76%). LC/MS RT=2.33 min.M/Z+323, 100%.

The β-carboline product/intermediate B (200 mg, 0.62 mmol) is thendissolved in 5 mL of dry THF and cooled to about 0° C. Lithium aluminumhydride (LAH) solution (1.2 mL, 1.0M in ether, 1.2 mmol) is added to thecooled reaction mixture under nitrogen. After the addition is complete(about 10 minutes), the reaction is allowed to warm to room temperaturefor about 4 hours. The reaction mixture is then cooled back to 0° C.,and saturated sodium sulfate solution (750 μL) is added and the mixturestirred for about 5 minutes at 0° C. The reaction mixture is thenfiltered and washed with THF (100 mL). The solvent is removed in vacuo,and the crude product purified by preparative HPLC. The product C([1-(3-Methoxy-phenyl)-2,3,4,9-tetrahydro-1H-b-carbolin-3-yl]-methanol)is isolated as a white solid (106 mg, 55%). LC/MS RT=2.25 min. M/Z+309,100%.

Example 1F Chemical Resolution of Compounds of the Invention

Compounds of the invention may optionally be chemical resolved toenantiomerically pure compositions, preferably enantiomerically pure (S)isomer compositions as follows.

The racemic amine A (18.21 g, 58.2 mmol) is mixed withN-acetyl-L-phenylalanine (12.05 g, 58.2 mmol) in EtOH (1.28 L) andrefluxed to get a clear solution. The solution is then allowed to coolto room temperature. After overnight standing, the precipitated solid isfiltered and washed with EtOH (200 mL) to give the salt B (16.4 g). Thesalt B is taken in EtOAc (500 mL) and washed with aqueous 1N NaOH (300mL×2) or NH₄OH (200 mL×2), dried and evaporated to give the S-isomer ofthe free amine C (7.4 g). The R-isomer is prepared by similar procedureusing N-acetyl-D-phenylalanine.

Example 1G Further Exemplary Compounds of the Invention

By way of further non-limiting example, the following compounds (Table5) may be prepared by similar methodology to that described above, aswill be recognized by one of skill in the art.

TABLE 5 Mass Reten. Spec Time Compound NMR (LCMS) (min)

(CDCl3, 400 MHz), δ 8.16 (s, 1 H), 7.48 (s, 1 H), 7.22 (d, J = 8.8 Hz, 1H), 7.19 (d, J = 8.8 Hz, 2 H), 7.13 (d, J = 8.8 Hz, 1 H), 6.94 (s, 1 H),6.80 (d, J = 8.8 Hz, 2 H), 3.92-3.91 (m, 1 H), 3.86 (t, J = 7.2 Hz, 2H), 3.77 (s, 3 H), 3.46-3.39 (m, 1 H), 3.11-3.09 (m, 1 H), 2.91-2.83 (m,3 H) 402.8 4.37

(CDCl3, 400 MHz), δ 8.29 (s, 1 H), 7.47-7.09 (m, 10 H), 6.98 (s, 1 H),6.77 (d, J = 8.8 Hz, 2 H), 3.93 (dd, J = 13.6 Hz and 4.8 Hz, 1 H), 3.82-3.80 (m, 2 H), 3.77 (s, 3 H), 3.38- 3.30 (m, 1 H), 2.69-2.65 (m, 1 H),2.53-2.45 (m, 1 H) 430.9 4.79

(CDCl3, 400 MHz), δ 8.21 (s, 1 H), 7.46 (s, 1 H), 7.22 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2 H), 7.12 (dd, J = 8.4 Hz and 2.0 Hz, 1 H),6.92 (s, 1 H), 6.77 (d, J = 8.4 Hz, 2 H), 3.94 (dd, J = 13.2 Hz and 4.4Hz, 1 H), 3.76 (s, 3 H), 3.65 (s, 3 H), 3.43-3.35 (m, 1 H), 2.87-2.62(m, 6 H) 427.0 4.06

(CDCl3, 400 MHz), δ 8.23, 8.12 (s, 1 H), 7.48, 7.42 (d, J = 1.6 Hz, 1.2Hz, 1 H), 7.22-7.10 (m, 4 H), 6.94, 6.88 (s, 1 H), 6.79 (d, J = 8.8 Hz,2 H), 5.48-5.45 (m, 1 H), 3.96-3.80 (m, 1 H), 3.77 (s, 3 H), 3.47-3.36(m, 1 H), 3.08-2.77 (m, 2 H), 2.14, 2.09 (s, 3 H), 1.48, 1.41 (d, J =6.8 Hz, 6.4 Hz, 3 H) 427.0 3.99

(CDCl3, 400 MHz), δ 7.87 (s, 1 H), 7.51 (s, 1 H), 7.47 (dd, J = 6.8 Hzand 1.6 Hz, 1 H), 7.30-7.15 (m, 6 H), 6.98 (b, 1 H), 6.76 (d, J = 8.8Hz, 2 H), 3.80 (s, 3 H), 3.77-3.74 (m, 1 H), 3.49-3.39 (m, 1 H),2.93-2.82 (m, 2 H) 469.0 5.27

(CDCl3, 400 MHz), δ 8.07 (dd, J = 7.6 Hz and 1.2 Hz, 1 H), 7.74 (s, 1H), 7.45-7.32 (m, 4 H), 7.18 (d, J = 8.4 Hz, 1 H), 7.12 (dd, J = 8.8 Hzand 2.0 Hz, 1 H), 7.07 (d, J = 8.4 Hz, 2 H), 6.76 (d, J = 8.8 Hz, 2 H),6.35 (s, 1 H), 3.97 (dd, J = 14.8 Hz and 5.2 Hz, 1 H), 3.77 (s, 3 H),3.49-3.41 (m, 1 H), 2.67 (dd, J = 15.6 Hz and 3.2 Hz, 1 H), 2.57-2.53(m, 1 H) 486.9 4.96

(CDCl3, 400 MHz), δ 7.95 (s, 1 H), 7.48 (s, 1 H), 7.30 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.8 Hz, 1 H), 7.16 (dd, J = 8.8 Hz and 1.6 Hz, 1 H),7.05 (b, 3 H), 6.86 (d, J = 8.4 Hz, 2 H), 3.80 (s, 3 H), 3.61 (dd, J =13.6 Hz and 5.2 Hz, 1 H), 3.52-3.44 (m, 1 H), 2.91-2.88 (m, 1 H), 2.78(dd, J = 15.2 Hz and 3.2 Hz, 1 H) 470.8 5.01

(CDCl3, 400 MHz), δ 8.09 (s, 1 H), 7.45 (s, 1 H), 7.21-7.17 (m, 4 H),7.12 (d, J = 8.8 Hz, 1 H), 6.98 (s, 1 H), 6.91 (d, J = 4 Hz, 1 H), 6.80(s, 1 H), 6.79 (d, J = 8.4 Hz, 2 H), 3.99 (s, 2 H), 3.96 (d, J = 4.4 Hz,1 H), 3.77 (s, 3 H), 3.43-3.38 (m, 1 H), 2.77-2.63 (m, 2 H) 436.9 4.66

(CDCl3, 400 MHz), δ 8.19, 8.16 (s, 1 H), 7.48, 8.42 (s, 1 H), 7.24-7.09(m, 6 H), 6.94 (t, J = 7.8 Hz, 2 H), 6.85 (t, J = 8.2 Hz, 2 H), 6.77 (d,J = 8.4 Hz, 1 H), 6.72 (d, J = 8.4 Hz, 1 H), 5.09-4.98 (m, 1 H),4.39-4.17 (m, 1 H), 3.77, 3.75 (s, 3 H), 3.41- 3.28 (m, 1 H), 3.02-2.65(m, 2 H), 1.61-1.59 (m, 3 H) 461 4.92

(CDCl3, 400 MHz), δ 8.39 (s, 1 H), 7.48 (s, 1 H), 7.23 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2 H), 7.13 (dd, J = 8.8 Hz and 1.6 Hz, 1 H),6.89 (s, 1 H), 6.77 (d, J = 8.4 Hz, 2 H), 4.17 (q, J = 12.8 Hz, 2 H),3.88 (d, J = 10 Hz, 1 H), 3.75 (s, 3 H), 3.41 (s, 3 H), 3.38-3.34 (m, 1H), 2.95-2.81 (m, 2 H) 385 3.79

(CD3OD, 400 MHz), δ 7.48-7.46 (m, 4 H), 7.35 (b, 1 H), 7.23 (d, J = 8.8Hz, 1 H), 7.07 (dd, J = 8.8 Hz and 2.0 Hz, 1 H), 6.46 (b, 1 H), 4.35-4.14 (m, 5 H), 3.52-3.47 (m, 2 H), 3.22-3.19 (m, 7 H), 2.98-2.93 (m, 3H), 2.89 (s, 6 H), 2.67-2.63 (m, 5 H), 2.06-1.96 (m, 2 H), 1.31 (t, J =7.2 Hz, 3 H) 538.3 4.29

(DMSO, 400 MHz), δ 11.00 (s, 1 H), 8.47 (s, 2 H), 7.67 (s, 1 H), 7.26(d, J = 8.4 Hz, 1 H), 7.19 (dd, J = 8.8 Hz and 2.0 Hz, 1 H), 6.26 (b, 1H), 4.25 (b, 1 H), 4.11 (t, J = 6.8 Hz, 2 H), 3.22-3.17 (m, 1 H),2.86-2.81 (m, 1 H), 2.77-2.66 (m, 1 H), 2.50 (b, 3 H), 1.21 (t, J = 6.8Hz, 3 H) 447.1 6.55

(CD3OD, 400 MHz), δ 8.43-8.41 (m, 4 H), 7.63 (d, J = 1.2 Hz, 1 H), 7.22(d, J = 8.8 Hz, 1 H), 7.19 (dd, J = 8.4 Hz and 1.6 Hz, 1 H), 7.04 (s, 1H), 6.67 (t, J = 4.8 Hz, 1 H), 5.01 (dd, J = 14.0 Hz and 3.6 Hz, 1 H),3.29-3.26 (m, 1 H), 3.21 (s, 6 H), 2.91-2.86 (m, 2 H) 450.1 5.48

(DMSO, 400 MHz), δ 11.15, 11.05 (b, 1 H), 7.53 (d, J = 1.6 Hz, 1 H),7.29 (d, J = 8.8 Hz, 1 H), 7.20-7.18 (m, 6 H), 7.06 (dd, J = 8.8 Hz and2 Hz, 1 H), 6.93 (d, J = 7.2 Hz, 2 H), 6.45-6.37 (m, 1 H), 4.30 (b, 1H), 3.72 (s, 3 H), 3.18 (b, 1 H), 2.82 (b, 2 H) 451.3 3.99

(CD3OD, 400 MHz), δ 10.98 (b, 1 H), 7.49 (d, J = 2.0 Hz, 1 H), 7.34-7.30 (m, 5 H), 7.25-7.21 (m, 1 H), 7.13 (dd, J = 8.8 Hz and 2.0 Hz, 1H), 4.81-4.79 (m, 1 H), 3.82-3.76 (m, 1 H), 3.54-3.49 (m, 1 H),3.11-3.07 (m, 2 H), 2.91-2.87 (m, 2 H), 2.59- 2.55 (m, 1 H), 2.24-2.20(m, 1 H) 311.1 4.39

(CD3OD, 400 MHz), δ 7.61 (s, 1 H), 7.46 (d, J = 8.0 Hz, 2 H), 7.38 (d, J= 8.0 Hz, 2 H), 7.19 (s, 2 H), 6.47 (s, 1 H), 4.32-4.19 (m, 5 H), 3.62(t, J = 3.9 Hz, 2 H), 3.42 (s, 1 H), 3.19- 3.10 (m, 3 H), 2.29-2.76 (m,2 H), 1.30 (s, 3 H) 486.6 3.45

(CD3OD, 400 MHz), δ 7.63 (s, 1 H), 7.49 (d, J = 8.4 Hz, 2 H), 7.42 (d, J= 8.4 Hz, 2 H), 7.19 (s, 2 H), 6.49 (b, 1 H), 4.34-4.19 (m, 4 H), 3.60(b, 4 H), 3.29-3.17 (m, 6 H), 2.89-2.75 (m, 2 H), 1.36 (t, J = 7.2 Hz, 3H), 1.30 (b, 3 H) 539.2 3.11

(CDCl3, 400 MHz), δ 8.56 (b, 1 H), 8.40 (b, 2 H), 7.68 (s, 1 H), 7.28(d, J = 2.0 Hz, 1 H), 7.14 (d, J = 8.4 Hz, 1 H), 7.00 (d, J = 8.2 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2 H), 6.48-6.38 (m, 1 H), 4.55-4.52 (m, 1 H),3.81-3.74 (m, 4 H), 3.24 (s, 6 H), 3.00-2.91 (m, 1 H), 2.88-2.84 (m, 1H) 522.2 5.05

(DMSO, 400 MHz), δ 11.00 (s, 1 H), 8.14 (s, 2 H), 7.64 (s, 1 H), 7.23(d, J = 8.4 Hz, 1 H), 7.18 (d, J = 8.8 Hz, 1 H), 6.14 (s, 1 H), 4.23 (b,1 H), 4.11-4.08 (m, 2 H), 3.14-3.10 (m, 1 H), 3.08 (s, 6 H), 2.81-2.77(m, 1 H), 2.70-2.66 (m, 1 H), 1.21 (t, J = 6.8 Hz, 3 H) 444.3 3.95

(CD3OD, 400 MHz), δ 7.79 (d, J = 8.4 Hz, 2 H), 7.63 (s, 1 H), 7.37 (d, J= 8.4 Hz, 2 H), 7.20 (s, 2 H), 6.51 (b, 1 H), 4.32-4.22 (m, 3 H), 3.54(s, 3 H), 3.36 (s, 2 H), 3.30 (s, 2 H), 3.21-3.11 (m, 1 H), 2.90-2.77(m, 2 H), 1.32 (s, 3 H) 500.1 4.35

(CDCl3, 400 MHz), δ 7.98, 7.81 (s, 1 H), 7.42 (s, 1 H), 7.21 (d, J = 8.4Hz, 1 H), 7.11 (d, J = 8.4 Hz, 1 H), 5.40-5.23 (m, 3 H), 4.55-4.35 (m, 1H), 4.20-4.11 (m, 2 H), 3.24- 3.13 (m, 1 H), 2.79-2.63 (m, 2 H), 2.22(d, J = 6.8 Hz, 2 H), 2.08 (b, 2 H), 1.89-1.81 (m, 2 H), 1.30 (b, 3 H),0.97 (b, 3 H) 361.2 5.95

(CD3OD, 400 MHz), δ 7.47 (d, J = 1.6 Hz, 1 H), 7.43 (d, J = 7.6 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2 H), 7.24 (d, J = 8.8 Hz, 1 H), 7.06 (dd, J =8.4 Hz and 1.6 Hz, 1 H), 6.49 (b, 1 H), 4.35-4.21 (m, 3 H), 3.83 (s, 4H), 3.19-3.10 (m, 1 H), 2.90-2.79 (m, 2 H), 1.57 (b, 6 H), 1.32 (s, 3 H)482.1 5.11

(CDCl3, 400 MHz), δ 8.48-8.09 (m, 1 H), 7.44-7.42 (m, 1 H), 7.24 (t, J =9 Hz, 1 H), 7.11-7.09 (m, 1 H), 5.59-5.40 (m, 1 H), 4.54-4.34 (m, 1 H),4.21-4.18 (m, 2 H), 3.23-3.13 (m, 1 H), 2.87-2.81 (m, 2 H), 2.76- 263(m, 1 H), 2.17 (s, 3 H), 2.12- 1.90 (m, 2 H), 1.42-1.24 (m, 6 H) 367.12.92

(CD3OD, 400 MHz), δ 8.62 (d, J = 4.4 Hz, 2 H), 8.59 (s, 2 H), 7.84 (s, 1H), 7.43-7.39 (m, 2 H), 7.24 (s, 1 H), 6.88 (t, J = 8.0 Hz, 1 H), 5.24-5.20 (m, 1 H), 3.47-3.44 (m, 1 H), 3.16 (s, 3 H), 3.11-3.05 (m, 2 H)436.2 5.25

(CDCl3, 400 MHz), δ 8.12 (s, 1 H), 7.45 (s, 1 H), 7.26 (d, J = 8 Hz, 2H), 7.18 (d, J = 8.8 Hz, 2 H), 7.14-7.12 (m, 4 H), 6.97 (s, 1 H), 6.78(d, J = 8.8 Hz, 2 H), 3.89 (dd, J = 14 Hz and 1.2 Hz, 1 H), 3.80-3.78(m, 5 H), 3.41-3.33 (m, 1 H), 2.73 (dd, J = 15.2 Hz and 3.2 Hz, 1 H),2.64- 2.60 (m, 1 H) 464.9 5.11

(CD3OD, 400 MHz), δ 7.78 (d, J = 8.0 Hz, 2 H), 7.47 (d, J = 1.6 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.06 (dd, J =8.8 Hz and 1.6 Hz, 1 H), 6.49 (b, 1 H), 4.31-4.05 (m, 8 H), 3.20-3.11(m, 1 H), 3.00-2.77 (m, 4 H), 1.94- 1.90 (m, 2 H), 1.54-1.45 (m, 2 H),1.31 (b, 3 H), 1.25 (t, J = 7.2 Hz, 3 H) 553.1 6.13

(CD3OD, 400 MHz), δ 7.80 (d, J = 8.0 Hz, 2 H), 7.48 (d, J = 1.6 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2 H), 7.25 (d, J = 8.8 Hz, 1 H), 7.07 (dd, J =8.4 Hz and 1.6 Hz, 1 H), 6.49 (b, 1 H), 4.31-4.21 (m, 4 H), 4.06 (t, J =8.4 Hz, 1 H), 3.74 (t, J = 8.0 Hz, 1 H), 3.51 (d, J = 5.2 Hz, 2 H),3.21- 3.11 (m, 1 H), 2.90-2.79 (m, 2 H), 2.26 (s, 1 H), 1.39 (s, 3 H),1.32 (s, 6 H) 454.3 5.98

(CDCl3, 400 MHz), δ 8.29 (b, 1 H), 7.64 (d, J = 8.0 Hz, 2 H), 7.61 (d, J= 7.2 Hz, 2 H), 7.50-7.45 (m, 5 H), 7.39 (d, J = 7.6 Hz, 1 H), 7.33 (d,J = 7.6 Hz, 2 H), 7.19 (d, J = 8.8 Hz, 1 H), 7.14 (dd, J = 8.4 Hz and1.6 Hz, 1 H), 7.08 (s, 1 H), 6.84 (d, J = 8 Hz, 2 H), 3.87 (d, J = 9.2Hz, 1 H), 3.79 (s, 3 H), 3.45-3.40 (m, 1 H), 2.96- 2.94 (m, 1 H),2.80-2.76 (m, 1 H) 493.0 5.71

(CD3OD, 400 MHz), δ 7.63 (s, 1 H), 7.48 (d, J = 8.4 Hz, 2 H), 7.42 (d, J= 8.0 Hz, 2 H), 7.20 (s, 2 H), 6.49 (b, 1 H), 4.33-4.22 (b, 3 H), 3.89(t, J = 5.2 Hz, 2 H), 3.50 (b, 4 H), 3.21- 3.11 (m, 2 H), 2.91-2.78 (m,2 H), 1.31 (s, 3 H) 555.2 3.14

(CD3OD, 400 MHz), δ 7.47 (d, J = 2.0 Hz, 1 H), 7.39 (s, 4 H), 7.23 (d, J= 8.8 Hz, 1 H), 7.06 (dd, J = 8.4 Hz and 2.0 Hz, 1 H), 6.49 (b, 1 H),4.35-4.21 (m, 3 H), 3.75 (b, 2 H), 3.53 (t, J = 5.4 Hz, 2 H), 3.44 (b, 2H), 3.26-3.30 (m, 4 H), 3.22-3.13 (m, 1 H), 2.89-2.78 (m, 2 H), 2.60 (t,J = 5.4 Hz, 4 H), 2.46 (b, 2 H), 1.32 (s, 3 H) 525.2 5.07

(CDCl3, 400 MHz), δ 7.80, 7.75 (s, 1 H), 7.43, 7.41 (s, 1 H), 7.21 (d, J= 8.4 Hz, 1 H), 7.10 (d, J = 8.0 Hz, 1 H), 5.43, 5.27 (d, J = 7.2 Hz, 1H), 4.51-4.30 (m, 1 H), 4.21-4.10 (m, 2 H), 3.18 (q, J = 12.8 Hz, 1 H),2.82- 2.76 (m, 1 H), 2.64-2.61 (m, 1 H), 1.82-1.76 (m, 2 H), 1.55-1.53(m, 1 H), 1.29-1.24 (m, 3 H), 1.08 (b, 3 H), 0.98 (d, J = 6.8 Hz, 3 H)335.3 5.52

(CD3OD, 400 MHz), δ 7.47 (d, J = 2.0 Hz, 1 H), 7.39 (s, 4 H), 7.23 (d, J= 8.8 Hz, 1 H), 7.05 (dd, J = 8.4 Hz and 2.0 Hz, 1 H), 6.49 (b, 1 H),4.32-4.20 (m, 3 H), 3.76 (b, 2 H), 3.46 (b, 2 H), 3.21-3.13 (m, 1 H),2.90-2.78 (m, 2 H), 2.54 (b, 2 H), 2.49-2.43 (m, 4 H), 1.32 (b, 3 H),1.10 (t, J = 7.2 Hz, 3 H) 495.3 4.68

((CD3OD, 400 MHz), δ 7.61 (s, 1 H), 7.44 (d, J = 8.0 Hz, 2 H), 7.35 (d,J = 8.0 Hz, 2 H), 7.20-7.16 (m, 2 H), 6.45 (b, 1 H), 4.28-4.14 (m, 3 H),4.11 (s, 2 H), 3.47 (s, 4 H), 3.26 (s, 4 H), 3.19-3.12 (m, 1 H), 2.91(s, 3 H), 2.88-2.79 (m, 2 H), 1.30 (s, 3 H) 511.2 4.99

(CD3OD, 400 MHz), δ 7.48 (d, J = 1.6 Hz, 1 H), 7.34 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 8.8 Hz and 2.0 Hz, 1 H), 4.68 (s, 1 H), 3.77-3.72 (m,1 H), 3.47-3.44 (m, 1 H), 3.10-3.03 (m, 2 H), 2.65-2.61 (m, 1 H), 1.25(d, J = 7.2 Hz, 3 H), 0.96 (d, J = 7.2 Hz, 3 H) 249.1 3.67

CD3OD, 400 MHz), δ 7.63 (s, 1 H), 7.48 (d, J = 8.0 Hz, 2 H), 7.42 (d, J= 8.0 Hz, 2 H), 7.20 (s, 2 H), 6.49 (b, 1 H), 4.32-4.21 (m, 3 H), 3.50(b, 4 H), 3.21-3.15 (m, 3 H), 2.92 (s, 3 H), 2.90-2.73 (m, 2 H), 1.32(s, 3 H) 525.1 3.25

(CD3OD, 400 MHz), δ 7.78 (d, J = 8.0 Hz, 2 H), 7.63 (s, 1 H), 7.37 (d, J= 8.4 Hz, 2 H), 7.20 (s, 2 H), 6.49 (b, 1 H), 4.31-4.22 (m, 3 H),3.19-3.11 (m, 1 H), 2.90 (s, 3 H), 2.86-2.77 (m, 2 H), 1.32 (s, 3 H)456.1 4.26

(CD3OD, 400 MHz), δ 7.48 (d, J = 2 Hz, 1 H), 7.41-7.36 (m, 4 H), 7.23(d, J = 8.4 Hz, 1 H), 7.06 (dd, J = 8.8 Hz and 2.0 Hz, 1 H), 6.49 (b, 1H), 4.35-4.21 (m, 3 H), 3.64 (b, 2 H), 3.45 (b, 2 H), 3.20-3.11 (m, 1H), 2.92-2.78 (m, 2 H), 2.68 (b, 2 H), 2.55 (b, 2 H), 1.92-1.80 (m, 4H), 1.66-1.62 (m, 1 H), 1.32-1.22 (m, 8 H) 549.3 5.29

(CD3OD, 400 MHz), δ 7.63 (s, 1 H), 7.41 (d, J = 8.4 Hz, 2 H), 7.37 (d, J= 8.0 Hz, 2 H), 7.19 (s, 2 H), 6.49 (b, 1 H), 4.35-4.22 (m, 3 H), 3.22-3.13 (m, 1 H), 3.08 (s, 3 H), 2.98 (s, 3 H), 2.89-2.77 (m, 2 H), 1.32(s, 3 H) 470.1 4.46

(CD3OD, 400 MHz), δ 7.63 (s, 1 H), 7.48 (d, J = 7.2 Hz, 2 H), 7.40 (d, J= 8.0 Hz, 2 H), 7.20 (s, 2 H), 6.49 (b, 1 H), 4.35-4.22 (m, 4 H), 3.82-3.50 (m, 6 H), 3.45 (b, 1 H), 3.21- 3.11 (m, 1 H), 3.00-2.78 (m, 5 H),2.25-2.15 (m, 2 H), 1.32 (s, 3 H) 539.2 3.02

(CDCl3, 400 MHz), δ 8.06 7.98 (s, 1 H), 7.50, 7.49 (s, 1 H), 7.22 (d, J= 6.0 Hz, 1 H), 7.21 (d, J = 6.4 Hz, 2 H), 7.15 (dd, J = 8.8 Hz and 1.6Hz, 1 H), 6.81 (d, J = 8.4 Hz, 2 H), 6.77 (s, 1 H), 3.91 (s, 3 H), 3.77(s, 3 H), 3.72 (d, J = 5.2 Hz, 1 H), 3.51-3.43 (m, 1 H), 3.02-2.96 (m, 1H), 2.86- 2.81 (m, 1 H) 398.9 4.18

(CDCl3, 400 MHz), δ 7.77, 7.70 (s, 1 H), 7.42, 7.39 (s, 1 H), 7.20 (dd,J = 8.4 Hz and 1.6 Hz, 1 H), 7.09 (d, J = 8.0 Hz, 1 H), 5.52-5.36 (m, 1H), 4.44-4.17 (m, 3 H), 3.28-3.20 (m, 1 H), 2.88-2.77 (m, 1 H), 2.60 (d,J = 15.2 Hz, 1 H), 2.05-1.88 (m, 1 H), 1.58-1.54 (m, 1 H), 1.30-1.26 (m,3 H), 1.04 (d, J = 2 Hz, 9 H) 349.1 6.03

(CD3OD, 400 MHz), δ 7.85 (d, J = 8.0 Hz, 2 H), 7.64 (s, 1 H), 7.41 (d, J= 8.4 Hz, 2 H), 7.20 (s, 2 H), 6.52 (b, 1 H), 4.33-4.22 (b, 3 H), 4.07(b, 2 H), 3.77 (t, J = 5.6 Hz, 4 H), 3.65 (b, 2 H), 3.39 (t, J = 5.6 Hz,2 H), 3.21-3.11 (m, 3 H), 2.91-2.78 (m, 2 H), 1.32 (s, 3 H) 555.2 3.34

(CD3OD, 400 MHz), δ 7.81 (d, J = 8.4 Hz, 2 H), 7.63 (s, 1 H), 7.37 (d, J= 8.0 Hz, 2 H), 7.20 (s, 2 H), 6.51 (b, 1 H), 4.32-4.22 (m, 3 H), 3.69(t, J = 5.8 Hz, 2 H), 3.48 (t, J = 5.6 Hz, 2 H), 3.21-3.11 (m, 1 H),2.90-2.77 (m, 2 H), 1.32 (s, 3 H) 486.1 3.80

(CD3OD, 400 MHz), δ 7.47 (s, 1 H), 7.41-7.38 (m, 4 H), 7.23 (d, J = 8.8Hz, 1 H), 7.06 (dd, J = 8.8 Hz and 1.6 Hz, 1 H), 6.49 (b, 1 H), 4.35-4.21 (m, 3 H), 3.73-3.62 (m, 6 H), 3.44 (b, 2 H), 3.19-3.10 (m, 1 H),2.91-2.78 (m, 2 H), 1.32 (b, 3 H) 468 5.52

(DMSO, 400 MHz), δ 11.19 (b, 1 H), 8.49 (b, 1 H), 7.81 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 1.6 Hz, 1 H), 7.30 (d, J = 8.4 Hz, 2 H), 7.29 (d, J =14.0 Hz, 1 H), 7.07 (dd, J = 8.4 Hz and 1.6 Hz, 1 H), 6.39 (b, 1 H),4.21- 4.16 (m, 3 H), 3.93 (t, J = 6.4 Hz, 1 H), 3.74 (q, J = 6.8 Hz, 1H), 3.59 (q, J = 6.8 Hz, 1 H), 3.28 (s, 2 H), 3.08-3.01 (m, 1 H),2.81-2.70 (m, 2 H), 1.91-1.79 (m, 3 H), 1.59-1.52 (m, 1 H), 1.21 (s, 3H) 482.2 5.74

(CD3OD, 400 MHz), δ 11.05 (s, 1 H), 8.09 (s, 2 H), 7.64 (s, 1 H), 7.32(b, 1 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.17 (dd, J = 8.8 Hz and 2.0 Hz, 1H), 6.24 (s, 1 H), 4.22 (b, 1 H), 4.12-4.09 (m, 2 H), 3.15-3.09 (m, 1H), 2.83-2.65 (m, 5 H), 1.21 (t, J = 6.8 Hz, 3 H) 430.2 3.65

(CD3OD, 400 MHz), δ 7.49 (d, J = 1.6 Hz, 1 H), 7.34 (d, J = 8.8 Hz, 1H), 7.13 (dd, J = 8.8 Hz and 2.0 Hz, 1 H), 3.77-3.72 (m, 1 H), 3.52-3.45(m, 1 H), 3.15-3.01 (m, 2 H), 2.80- 2.74 (m, 2 H), 2.60-2.52 (m, 1 H),2.27-2.20 (m, 4 H) 281.0 3.84

(CDCl3, 400 MHz), δ 8.35 (b, 1 H), 7.51 (s, 1 H), 7.32-7.26 (m, 4 H),7.20 (d, J = 8.4 Hz, 1 H), 7.13 (dd, J = 8.8 Hz and 2.4 Hz, 1 H), 6.39(b, 1 H), 4.25-4.21 (m, 2 H), 3.80 (b, 2 H), 3.47 (b, 2 H), 3.16-3.10(m, 1 H), 2.96-2.88 (m, 3 H), 2.79-2.75 (m, 1 H), 2.54-2.36 (m, 6 H),1.32 (s, 3 H) 481.4 4.81

(DMSO, 400 MHz), δ 10.86 (s, 1 H), 8.17 (s, 1 H), 8.03 (d, J = 7.6 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1 H), 7.65 (d, J = 8.4 Hz, 1 H), 7.50 (b, 2 H),7.26 (d, J = 8.4 Hz, 1 H), 7.02 (d, J = 8.8 Hz, 1 H), 6.24 (s, 1 H),4.35 (b, 1 H), 4.09-4.05 (m, 2 H), 3.61- 3.49 (m, 1 H), 2.78-2.65 (m, 2H), 1.45 (t, J = 6.8 Hz, 3 H) 423.3 5.15

(CD3OD, 400 MHz), δ 8.33 (s, 2 H), 7.67 (s, 1 H), 7.23 (s, 2 H), 7.05(d, J = 8.4 Hz, 2 H), 6.91 (d, J = 8.8 Hz, 2 H), 6.54-6.38 (m, 1 H),4.52 (b, 1 H), 3.78 (s, 3 H), 3.36-3.34 (m, 1 H), 2.99 (s, 3 H),2.92-2.88 (m, 2 H) 508.2 5.72

(CDCl3, 400 MHz), δ 7.88-7.77 (m, 1 H), 7.43 (d, J = 8.0 Hz, 1 H), 7.23(d, J = 8.8 Hz, 1 H), 7.11 (d, J = 8.8 Hz 1 H), 5.70-7.68 (m, 2 H),5.19-4.97 (m, 1 H), 4.60-4.38 (m, 1 H), 4.19- 4.07 (m, 2 H), 2.82-2.80(m, 1 H), 2.68-2.64 (m, 1 H), 2.29-1.84 (m, 6 H), 1.55-1.46 (m, 1 H),1.36-1.24 (m, 3 H) 359.1 5.65

(CD3OD, 400 MHz), δ 7.84 (d, J = 8.0 Hz, 2 H), 7.63 (s, 1 H), 7.38 (d, J= 8.0 Hz, 2 H), 7.20 (s, 2 H), 6.49 (b, 1 H), 4.31-4.22 (m, 3 H),3.19-3.11 (m, 1 H), 2.89-2.77 (m, 2 H), 1.32 (s, 3 H) 442.0 4.06

(CD3OD, 400 MHz), δ 8.44 (s, 2 H), 7.67 (d, J = 2.0 Hz, 1 H), 7.44 (d, J= 8.8 Hz, 1 H), 7.28 (dd, J = 8.8 Hz and 2.0 Hz, 1 H), 6.52 (s, 1 H),4.58- 4.55 (m, 1 H), 4.43-4.40 (m, 2 H), 3.41-3.31 (m, 1 H), 3.15 (s, 3H), 3.03-3.01 (m, 2 H), 1.32 (b, 3 H) 386.3 5.32

(CDCl3, 400 MHz), δ 7.66 (d, J = 24.8 Hz, 1 H), 7.39-6.89 (m, 8 H),5.44-5.02 (m, 1 H), 4.49-4.10 (m, 3 H), 3.23-2.94 (m, 2 H), 2.83-2.74(m, 1 H), 2.64-2.58 (m, 1 H), 2.26- 1.98 (m, 2 H), 1.47-1.26 (m, 6 H)397.1 5.97

(CD3OD, 400 MHz), δ 7.80 (d, J = 8.4 Hz, 2 H), 7.47 (d, J = 1.6 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2 H), 7.24 (d, J = 8.8 Hz, 1 H), 7.07 (dd, J =8.0 Hz and 1.6 Hz, 1 H), 6.49 (b, 1 H), 4.35-4.21 (m, 3 H), 3.69 (t, J =4.6 Hz, 4 H), 3.53 (t, J = 6.8 Hz, 2 H), 3.19-3.10 (m, 1 H), 2.90-2.78(m, 2 H), 2.59 (t, J = 6.6 Hz, 4 H), 2.53 (s, 2 H), 1.32 (s, 3 H) 511.45.05

(CDCl3, 400 MHz), δ 8.09, 7.83 (s, 1 H), 7.42 (s, 1 H), 7.21 (d, J = 8.4Hz, 1 H), 7.09 (dd, J = 8.4 Hz and 1.2 Hz, 1 H), 5.33-5.21 (m, 1 H),4.50-4.34 (m,1 H), 4.21-4.10 (m, 2 H), 3.19-3.17 (m, 1 H), 2.77-2.74 (m,1 H), 2.67-2.61 (m, 1 H), 1.81 (s, 2 H), 1.52 (s, 2 H), 1.29-1.23 (m, 3H), 0.96 (s, 3 H) 321.4 5.19

(CDCl3, 400 MHz), δ 7.73-7.52 (m, 1 H), 7.47 (s, 1 H), 7.42-7.18 (m, 6H), 7.09 (dd, J = 8.8 Hz and 2.0 Hz, 1 H), 5.41-5.26 (m, 1 H), 4.56-4.32(m, 1 H), 4.23-4.10 (m, 2 H), 3.21 (b, 1 H), 2.85-2.72 (m, 3 H), 2.65(d, J = 14.2 Hz, 1 H), 2.23-2.10 (m, 2 H), 1.38 (b, 3 H) 383.1 5.75

(CD3OD, 400 MHz), δ 7.80 (d, J = 8.4 Hz, 2 H), 7.47 (d, J = 1.6 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2 H), 7.24 (d, J = 8.8 Hz, 1 H), 7.06 (dd, J =8.8 Hz and 2.0 Hz, 1 H), 6.50 (b, 1 H), 4.32-4.21 (m, 3 H), 3.47 (t, J =7.2 Hz, 2 H), 3.38-3.34 (m, 4 H), 3.19-3.10 (m, 1 H), 2.89-2.78 (m, 2H), 2.39 (t, J = 8.4 Hz, 2 H), 2.09- 2.00 (m, 2 H), 1.86-1.80 (m, 2 H),1.32 (b, 3 H) 523.1 5.69

(CDCl3, 400 MHz), δ 7.81 (s, 1 H), 7.51 (d, J = 6.8 Hz, 1 H), 7.29 (dd,J = 12.0 Hz and 2.8 Hz, 1 H), 7.21 (d, J = 8.4 Hz, 1 H), 7.13 (dd, J =8.4 Hz and 2.0 Hz, 1 H), 7.12-7.08 (m, 1 H), 7.07 (s, 1 H), 6.50 (b, 1H), 4.49- 4.21 (m, 3 H), 3.17-3.09 (m, 1 H), 2.91-2.85 (m, 1 H),2.77-2.73 (m, 1 H), 1.39 (s, 3 H) 361.1 5.12

(CD3OD, 400 MHz), δ 7.47 (d, J = 2.0 Hz, 1 H), 7.46-7.37 (m, 4 H), 7.23(d, J = 8.4 Hz, 1 H), 7.06 (dd, J = 8.8 Hz and 2.0 Hz, 1 H), 6.49 (b, 1H), 4.35-4.21 (m, 3 H), 3.77-3.69 (m, 2 H), 3.55-3.45 (m, 2 H), 3.20-3.11 (m, 1 H), 2.90-2.78 (m, 3 H), 2.67-2.55 (m, 3 H), 2.39-2.31 (m, 3H), 2.01-1.95 (m, 1 H), 1.82-1.79 (m, 1 H), 1.32 (s, 3 H) 495.3 4.67

(CDCl3, 400 MHz), δ 7.92, 7.82 (s, 1 H), 7.42 (s, 1 H), 7.22 (dd, J =8.4 Hz and 1.2 Hz, 1 H), 7.10 (d, J = 8.8 Hz, 1 H), 5.31, 5.19 (s, 1 H),4.52, 4.32 (d, J = 10.8 Hz, 1 H), 4.20- 4.12 (m, 2 H), 3.19-3.12 (m, 1H), 2.81-2.62 (m, 2 H), 1.81 (d, J = 6.8 Hz, 2 H), 1.48-22 (m, 12 H),0.88 (s, 3 H) 363.5 6.34

(CD3OD, 400 MHz), δ 7.63 (s, 1 H), 7.50 (d, J = 8.0 Hz, 2 H), 7.40 (d, J= 8.0 Hz, 2 H), 7.20 (s, 2 H), 6.49 (b, 1 H), 4.30-4.20 (m, 3 H), 3.89(s, 2 H), 3.45 (b, 2 H), 3.20-3.10 (m, 1 H), 3.03-3.01 (m, 9 H),2.91-2.80 (m, 2 H), 1.32 (s, 3 H) 527.1 3.16

(CD3OD, 400 MHz), δ 8.27 (s, 2 H), 7.52 (d, J = 2 Hz, 1 H), 7.27 (d, J =8.8 Hz, 1 H), 7.17 (d, J = 8.8 Hz, 1 H), 7.10 (dd, J = 8.8 Hz and 2.4Hz, 1 H), 7.05 (d, J = 8.8 Hz, 2 H), 6.95 (d, J = 9.2 Hz, 2 H), 6.92 (s,1 H), 6.58-6.38 (m, 1 H), 4.52 (b, 1 H), 3.80 (s, 1 H), 3.79 (s, 3 H),3.31- 3.30 (m, 1 H), 2.95 (s, 3 H), 2.92- 2.88 (m, 1 H) 464.2 5.86

(CD3OD, 400 MHz), δ 8.49, 8.29 (d, J = 4.4 Hz, 2.8 Hz, 1 H), 7.82, 7.70(t, J = 2.0 Hz, 1 H), 7.46 (s, 1 H), 7.38-7.23 (m, 5 H), 7.15 (d, J =7.6 Hz, 1 H), 7.07 (d, J = 8.4 Hz, 1 H), 6.98 (d, J = 6.8 Hz, 1 H), 6.46(b, 1 H), 4.35-4.21 (m, 3 H), 3.88 (t, J = 7.0 Hz, 1 H), 3.71-3.67 (m, 1H), 3.20-3.11 (m, 3 H), 3.01-2.80 (m, 4 H), 1.32 (s, 3 H) 517.6 5.03

(DMSO, 400 MHz), δ 11.15 (s, 1 H), 7.51 (d, J = 2.0 Hz, 1 H), 7.42 (t, J= 7.6 Hz, 1 H), 7.35 (d, J = 7.6 Hz, 1 H), 7.30 (d, J = 8.8 Hz, 2 H),7.16 (s, 1 H), 7.06 (dd, J = 8.4 Hz and 2.0 Hz, 1 H), 6.36 (b, 1 H),4.18-4.10 (m, 3 H), 3.09-3.00 (m, 1 H), 2.91- 2.64 (m, 8 H), 1.21 (t, J= 6.6 Hz, 3 H) 426.2 4.29

(CD3OD, 400 MHz), δ 7.81 (d, J = 8.4 Hz, 2 H), 7.47 (d, J = 1.6 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.07 (dd, J =8.4 Hz and 2.0 Hz, 1 H), 6.50 (b, 1 H), 4.35-4.29 (m, 3 H), 3.70-3.60(m, 1 H), 3.51-3.47 (m, 2 H), 3.37- 3.29 (m, 1 H), 3.19-3.11 (m, 2 H),2.92 (s, 3 H), 2.88-2.78 (m, 2 H), 2.51-2.41 (m, 1 H), 2.29-2.20 (m, 1H), 2.17-2.00 (m, 2 H), 1.89-1.78 (m, 2 H), 1.32 (s, 3 H) 509.4 4.99

(CDCl3, 400 MHz), δ 7.91, 7.72 (s, 1 H), 7.50-7.43 (s, 1 H), 7.22-7.06(m, 6 H), 5.28-5.19 (m, 1 H), 4.64- 4.45 (m, 1 H), 4.20 (b, 2 H), 3.27-3.10 (m, 2 H), 2.91-2.72 (m, 2 H), 2.70-2.66 (m, 1 H), 2.49-2.28 (m, 2H), 1.38-1.24 (m, 9 H), 1.01, 0.96 (d, J = 6.8 Hz, 3 H) 439.0 6.11

(DMSO, 400 MHz), δ 11.10 (s, 1 H), 8.42 (s, 1 H), 7.75 (d, J = 7.2 Hz, 1H), 7.67 (s, 1 H), 7.51 (d, J = 1.6 Hz, 1 H), 7.43 (t, J = 7.2 Hz, 1 H),7.35 (d, J = 8.0 Hz, 1 H), 7.29 (d, J = 8.4 Hz, 1 H), 7.06 (dd, J = 8.8Hz and 2.4 Hz, 1 H), 6.39 (b, 1 H), 4.13-4.09 (m, 3 H), 3.10-3.04 (m, 1H), 2.81-2.72 (m, 5 H), 1.21 (s, 3 H) 412.1 4.13

(CD3OD, 400 MHz), δ 7.53-7.46 (m, 4 H), 7.29 (b, 1 H), 7.25 (d, J = 8.8Hz, 1 H), 7.08 (dd, J = 8.8 Hz and 2.08 Hz, 1 H), 6.49 (b, 1 H), 4.34-4.23 (m, 3 H), 3.53-3.42 (m, 2 H), 3.18-3.12 (m, 5 H), 2.91-2.74 (m, 3H), 1.32 (t, J = 7.2 Hz, 6 H) 4.95.3 3.46

(CD3OD, 400 MHz), δ 7.63 (s, 1 H), 7.51 (d, J = 8.0 Hz, 2 H), 7.41 (d, J= 8.4 Hz, 2 H), 7.19 (d, J = 1.2 Hz, 2 H), 6.46 (b, 1 H), 4.31 (s, 2 H),4.23-4.20 (m, 3 H), 3.62-3.50 (m, 4 H), 3.19-3.11 (m, 1 H), 2.92 (s, 6H), 2.87-2.81 (m, 2 H), 2.76 (s, 3 H), 1.31 (s, 3 H) 513.2 4.43

(CD3OD, 400 MHz), δ 7.47 (d, J = 2 Hz, 1 H), 7.46-7.37 (m, 4 H), 7.24(d, J = 8.8 Hz, 1 H), 7.07 (d, J = 8.8 Hz and 2.0 Hz, 1 H), 6.49 (b, 1H), 4.75 (b, 1 H), 4.35-4.21 (m, 3 H), 3.85 (b, 1 H), 3.64 (b, 2 H),3.45-3.37 (m, 1 H), 3.19-3.12 (m, 4 H), 2.91-2.80 (m, 3 H), 2.28-2.00(m, 6 H), 2.12-2.05 (m, 2 H), 1.61 (b, 2 H), 1.32 (s, 3 H) 535.3 4.94

(CDCl3, 400 MHz), δ 7.89-7.69 (m, 1 H), 7.43 (b, 1 H), 7.33-7.30 (m, 2H), 7.20-7.06 (m, 4 H), 5.29-5.19 (m, 1 H), 4.64-4.45 (m, 1 H), 4.20 (b,2 H), 3.27-3.10 (m, 2 H), 2.91- 2.72 (m, 2 H), 2.70-2.66 (m, 1 H), 2.50(b, 2 H), 2.29 (b, 1 H), 1.32- 1.31 (m, 12 H), 1.02, 0.90 (d, J = 6.8Hz, 3 H) 453.0 6.30

(CD3OD, 400 MHz), δ 7.52-7.45 (m, 4 H), 7.31 (b, 1 H), 7.25 (d, J = 8.4Hz, 1 H), 7.08 (dd, J = 8.4 Hz and 2.0 Hz, 1 H), 6.48 (b, 1 H), 4.34-4.23 (m, 3 H), 3.45 (b, 3 H), 3.23- 3.13 (m, 4 H), 2.92-2.80 (m, 5 H),1.32 (s, 3 H) 481.3 3.43

(CD3OD, 400 MHz), δ 7.48 (d, J = 1.6 Hz, 1 H), 7.43 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.07 (dd, J =8.4 Hz and 2.0 Hz, 1 H), 6.50 (b, 1 H), 4.35-4.29 (m, 3 H), 3.90 (b, 1H), 3.52-3.47 (m, 3 H), 3.20-3.16 (m, 2 H), 3.01 (t, J = 12.0 Hz, 2 H),2.91-2.79 (m, 3 H), 2.20 (b, 1 H), 2.00-1.97 (m, 3 H), 1.82-1.71 (m, 6H), 1.56-1.48 (m, 1 H), 1.32 (b, 3 H) 549.6 5.21

(DMSO, 400 MHz), δ 11.39 (s, 1 H), 9.80 (b, 1 H), 9.40 (b, 1 H), 7.52(d, J = 1.6 Hz, 1 H), 7.48 (s, 1 H), 7.37- 7.31 (m, 4 H), 7.25-7.19 (m,1 H), 7.00 (dd, J = 8.8 Hz and 2 Hz, 1 H), 4.76 (d, J = 5.6 Hz, 1 H),3.61-3.53 (m, 1 H), 3.25-3.20 (m, 1 H), 2.94- 2.92 (m, 2 H), 2.13-1.97(m, 1 H), 1.35, 1.24 (d, J = 6.8 Hz, 3 H) 325.3 4.75

(CD3OD, 400 MHz), δ 8.99 (s, 1 H), 7.80 (d, J = 8.0 Hz, 2 H), 7.71 (d, J= 1.2 Hz, 1 H), 7.57 (s, 1 H), 7.47 (d, J = 1.6 Hz, 1 H), 7.39 (d, J =8.0 Hz, 2 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.07 (d, J = 8.0 Hz, 1 H), 6.51(b, 1 H), 4.32 (t, J = 4.8 Hz, 3 H), 4.23-4.21 (m, 2 H), 3.43 (t, J =6.4 Hz, 2 H), 3.20-3.11 (m, 1 H), 2.91-2.78 (m, 2 H), 2.23-2.17 (m, 2H), 1.32 (b, 3 H) 506.2 4.96

(CD3OD, 400 MHz), δ 7.79 (d, J = 8.4 Hz, 2 H), 7.48 (s, 1 H), 7.38 (d, J= 8.0 Hz, 2 H), 7.25 (d, J = 8.8 Hz, 1 H), 7.07 (dd, J = 8.4 Hz and 2.0Hz, 1 H), 6.51 (b, 1 H), 4.35- 4.21 (m, 3 H), 3.67 (t, J = 4.6 Hz, 4 H),3.41 (q, J = 4.8 Hz, 2 H), 3.20- 3.11 (m, 1 H), 2.91-2.79 (m, 2 H), 2.62(s, 1 H), 2.46-2.42 (m, 5 H), 1.83-1.79 (m, 2 H), 1.32 (s, 3 H) 525.24.76

(CD3OD, 400 MHz), δ 7.62 (s, 1 H), 7.49 (d, J = 8.0 Hz, 2 H), 7.39 (d, J= 8.4 Hz, 2 H), 7.19 (s, 2 H), 6.48 (s, 1 H), 4.27-4.18 (m, 5 H), 3.87(t, J = 4.6 Hz, 4 H), 3.47 (t, J = 6.8 Hz, 2 H), 3.34-3.30 (m, 2 H),3.16-3.12 (m, 5 H), 2.89-2.75 (m, 2 H), 1.30 (s, 3 H) 541.2 3.51

(CD3OD, 400 MHz), δ 7.60 (s, 1 H), 7.51 (d, J = 8.0 Hz, 2 H), 7.40 (d, J= 8.0 Hz, 2 H), 7.21-7.16 (m, 2 H), 6.46 (b, 1 H), 4.41 (s, 2 H), 4.28-4.19 (m, 3 H), 3.79-3.74 (m, 4 H), 3.51-3.49 (m, 4 H), 3.19-3.11 (m, 1H), 2.95 (s, 3 H), 2.88-2.75 (m, 2 H), 2.30 (s, 2 H), 1.30 (s, 3 H)525.2 4.42

(CD3OD, 400 MHz), δ 7.84 (d, J = 8.0 Hz, 2 H), 7.47 (d, J = 2.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.06 (dd, J =8.4 Hz and 2.0 Hz, 1 H), 6.49 (b, 1 H), 4.35-4.16 (m, 3 H), 3.21-3.10(m, 1 H), 2.90-2.71 (m, 2 H), 1.32 (b, 3 H) 398.1 3.95

(CDCl3, 400 MHz), δ 7.92-7.77 (m, 1 H), 7.42-7.39 (m, 8 H), 7.26-7.21(m, 1 H), 7.10 (d, J = 8.4 Hz, 1 H), 5.16-4.97 (m, 1 H), 4.56-4.36 (m, 1H), 4.19-4.11 (m, 2 H), 3.27-3.19 (m, 1 H), 2.78-2.63 (m, 2 H), 1.90 (d,J = 5.6 Hz, 1 H), 1.74 (b, 1 H), 1.49-1.26 (m, 4 H), 1.10-0.91 (m, 6 H)335.2 5.45

(CD3OD, 400 MHz), δ 7.82 (s, 1 H), 7.80 (s, 1 H), 7.55-7.48 (m, 3 H),7.23 (d, J = 8.4 Hz, 1 H), 7.07 (dd, J = 8.4 Hz and 2.0 Hz, 1 H), 6.49(b, 1 H), 4.33-4.21 (m, 3 H), 4.05 (b, 2 H), 3.5-3.73 (m, 4 H), 3.61 (b,2 H), 3.37 (t, J = 5.8 Hz, 2 H), 3.25- 3.17 (m, 3 H), 2.92-2.80 (m, 2H), 1.32 (s, 3 H) 511.3 3.56

(CDCl3, 400 MHz), δ 8.01, 7.91 (s, 1 H), 7.43 (s, 1 H), 7.23 (d, J = 8.4Hz, 1 H), 7.11 (d, J = 7.2 Hz, 1 H), 6.71 (d, J = 7.6 Hz, 1 H), 6.63 (s,1 H), 6.57 (d, J = 7.6 Hz, 1 H), 5.92 (s, 2 H), 5.18-5.07 (m, 1 H),4.63-4.41 (m, 1 H), 4.30-4.11 (m, 2 H), 3.36-3.31 (m, 1 H), 2.91-2.83(m, 2 H), 2.70-2.61 (m, 1 H), 2.38- 2.15 (m, 2 H), 1.38-1.30 (m, 3 H),1.09-1.01 (m, 3 H) 440.9 5.75

(CD3OD, 400 MHz), δ 7.76 (s, 1 H), 7.75 (s, 1 H), 7.52-7.43 (m, 2 H),7.23 (d, J = 8.4 Hz, 1 H), 7.06 (d, J = 7.6 Hz, 1 H), 6.47 (b, 1 H),4.30- 4.21 (m, 3 H), 3.52 (s, 4 H), 3.33 (s, 3 H), 3.26-3.18 (m, 1 H),2.91-2.80 (m, 2 H), 1.32 (s, 3 H) 456.1 4.21

(CD3OD, 400 MHz), δ 7.48 (s, 1 H), 7.46 (d, J = 8.8 Hz, 2 H), 7.40 (d, J= 7.6 Hz, 2 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.07 (d, J = 8.0 Hz, 1 H),6.49 (b, 1 H), 4.35-4.21 (m, 3 H), 3.64- 3.61 (m, 2 H), 3.20-3.11 (m, 3H), 3.01 (s, 3 H), 2.93 (s, 5 H), 2.89- 2.78 (m, 3 H), 2.12-2.05 (m, 2H), 1.32 (s, 3 H) 497.2 4.69

(CDCl3, 400 MHz), δ 8.17, 8.00 (s, 1 H), 7.50 (s, 1 H), 7.23-7.13 (m, 4H), 6.97, 6.92 (s, 1 H), 6.80 (d, J = 8.4 Hz, 2 H), 4.43, 4.34 (t, J =7.0 Hz, 1 H), 4.04-3.98 (m, 1 H), 3.77 (s, 3 H), 3.47-3.41 (m, 1 H),3.25-2.81 (m, 2 H), 2.23-2.06 (m, 2 H), 1.02 (t, J = 6.2 Hz, 3 H) 460.84.96

(DMSO, 300 MHz), δ 7.63 (s, 1 H), 7.49 (d, J = 6.3 Hz, 2 H), 7.42 (d, J= 6.0 Hz, 2 H), 7.20 (s, 2 H), 6.49 (s, 1 H), 4.32-4.21 (m, 3 H), 3.85(b, 4 H), 3.39-3.30 (m, 3 H), 3.26-3.15 (m, 5 H), 2.92-2.73 (m, 9 H),2.26- 2.20 (m, 2 H), 1.31 (s, 3 H) 596.3 4.45

(CD3OD, 400 MHz), δ 7.52 (d, J = 8.4 Hz, 2 H), 7.47 (s, 1 H), 7.39- 7.36(m, 2 H), 7.24 (d, J = 8.8 Hz, 1 H), 7.06 (dd, J = 8.4 Hz and 1.6 Hz, 1H), 6.49 (b, 1 H), 4.45-4.23 (m, 4 H), 3.84-3.45 (m, 4 H), 3.20-3.12 (m,1 H), 2.91-2.78 (m, 2 H), 2.25- 2.10 (m, 1 H), 1.98-1.89 (m, 4 H), 1.32(s, 3 H) 509.2 5.18

(CD3OD, 400 MHz), δ 7.52-7.45 (m, 4 H), 7.32 (b, 1 H), 7.25 (d, J = 8.4Hz, 1 H), 7.08 (dd, J = 8.4 Hz and 1.6 Hz, 1 H), 6.49 (b, 1 H), 4.34-4.23 (m, 4 H), 3.69 (s, 3 H), 3.31- 3.30 (m, 8 H), 3.21-3.12 (m, 3 H),2.91-2.74 (m, 2 H), 1.32 (s, 3 H) 525.3 3.52

(CD3OD, 400 MHz), δ 7.51-7.48 (m, 3 H), 7.40 (d, J = 8.0 Hz, 2 H), 7.24(d, J = 8.8 Hz, 1 H), 7.07 (dd, J = 8.4 Hz and 1.2 Hz, 1 H), 6.49 (b, 1H), 4.35-4.21 (m, 3 H), 3.89 (b, 2 H), 3.45 (b, 2 H), 3.19-3.10 (m, 1H), 3.05-3.01 (m, 9 H), 2.91-2.78 (m, 2 H), 1.32 (b, 3 H) 483.1 4.96

(CD3OD, 400 MHz), δ 7.47 (d, J = 1.6 Hz 2 H), 7.35 (d, J = 8.4 Hz, 1 H),7.12 (dd, J = 8.4 Hz and J = 2.0 Hz, 1 H), 4.87 (s, 1 H), 3.75- 3.72 (m,1 H), 3.50-3.47 (m, 1 H), 3.09-3.03 (m, 2 H), 2.22 (dd, J = 15.6 Hz andJ = 2.4 Hz, 1 H), 1.84 (dd, J = 15.6 Hz and 8.4 Hz, 1 H), 1.17 (s, 9 H)276.9 4.00

(CD3OD, 400 MHz), δ 7.48 (d, J = 1.6 Hz, 1 H), 7.41-7.32 (m, 3 H), 7.23(d, J = 8.4 Hz, 2 H), 7.07 (dd, J = 8.4 Hz and 2.0 Hz, 1 H), 6.46 (b, 1H), 4.32-4.17 (m, 3 H), 3.80 (s, 2 H), 3.67 (t, J = 5.0 Hz, 2 H), 3.39(s, 3 H), 3.30-15 (m, 6 H), 2.88-2.83 (m, 6 H), 1.32 (s, 3 H) 511.4 4.71

(DMSO, 400 MHz), δ 11.39 (d, J = 2.8 Hz, 1 H), 9.75 (s, 1 H), 9.34 (s, 1H), 7.53 (s, 1 H), 7.36 (dd, J = 8.4 Hz and 4.0 Hz, 1 H), 7.10 (dd, J =8.8 Hz and 2.0 Hz, 1 H), 4.82- 4.71 (m, 1 H), 3.62-3.56 (m, 1 H), 3.14(b, 1 H), 3.00-2.83 (m, 2 H), 2.35-2.23 (m, 1 H), 2.18-1.82 (m, 4 H),1.34 (q, J = 6.4 Hz, 3 H) 295.0 4.14

Example 2 Assay to Evaluate Affect on Hypoxia-Inducible Endogenous VEGFExpression

The ability of the compounds of the invention to modulatehypoxia-inducible endogenous VEGF expression may be analyzed as follows.VEGF protein levels may be monitored by an ELISA assay (R&D Systems).Briefly, HeLa cells may be cultured for 24-48 hours under hypoxicconditions (1% O₂, 5% CO₂, balanced with nitrogen) in the presence orabsence of a compound of the invention. The conditioned media may thenbe assayed by ELISA, and the concentration of VEGF calculated from thestandard ELISA curve of each assay.

A dose-response analysis may be performed using the ELISA assay andconditions described above. The conditions for the dose-response ELISAare analogous to those described above. A series of, e.g., sevendifferent concentrations may be analyzed. In parallel, a dose-responsecytotoxicity assay may be performed using Cell Titer Glo (Promega) underthe same conditions as the ELISA to ensure that the inhibition of VEGFexpression was not due to the cytotoxicity. Dose-response curves may beplotted using percentage inhibition versus concentration of thecompound, and EC₅₀ and CC₅₀ values may be generated for each compoundwith the maximal inhibition set as 100% and the minimal inhibition as0%. Preferred compounds of the invention will have an EC₅₀ of less than50, preferably less than 10, more preferably less than 2, even morepreferably less than 0.5, and even more preferably less than 0.01.

FIG. 1 shows the ability of a typical compound of the invention,Compound No. 7, to inhibit endogenous VEGF production in tumor cellsunder hypoxic conditions. The ELISA EC₅₀ is 0.0025 μM, while its CC₅₀(50% cytotoxicity) is greater than 0.2 μM. The EC₅₀ for a series ofpreferred compounds of the invention is provided in Table 5.

TABLE 5 LCMS Retention ELISA Compound LCMS [M + H] Time (min) EC50 μM  1391.20 3.67 ****  2 385.28 4.01 *****  3 479.18 4.35 *****  4 435.234.28 *****  5 391.28 4.05 *****  6 425.28 4.07 *****  7 443.28 4.61*****  # 8 415.26 4.25 *****  9 431.25 4.07 ***** # 10   467.15 4.51*****  11 389.24 4.24 *****  12 414.31 3.94 *****  13 411.24 4.89 ***** 14 397.22 4.57 *****  15 457.3 4.24 *****  16 435.19 4.47 *****  17447.14 4.44 *****  18 431.14 4.55 *****  19 437.26 4.54 *****  20 389.244.22 *****  21 391.28 4.04 *****  22 425.28 4.11 *****  23 373.23 4.04*****  24 411.24 4.8 *****  25 449.23 4.03 *****  26 437.15 4.52 ***** 27 399.25 4.11 *****  28 399.19 4.2 *****  29 435.09 4.14 *****  30413.22 4.42 *****  31 423.17 4.32 *****  32 467.25 4.26 *****  33 457.154.29 *****  34 383.19 4.42 *****  35 425.28 4.14 *****  36 383.2 4.37*****  37 423.3 4.24 *****  38 355.24 4.07 *****  39 391.28 4.12 ***** 40 403.15 4.45 *****  41 449.11 4.59 *****  42 383.19 4.44 *****  43371.31 3.89 *****  44 479.18 4.35 *****  45 394.16 4.09 *****  46 421.194.22 ****  47 449.07 4.54 ****  48 403.32 4.2 ****  49 403.15 4.51 **** 50 405.18 3.81 ****  51 373.23 4.11 ****  52 355.3 4.07 ****  53 375.263.92 ****  54 435.23 4.3 ****  55 425.27 4.26 ****  56 414.14 4.19 **** 57 399.19 4.2 ****  58 469.22 4.32 ****  59 444.12 4.12 ****  60 433.174.27 ****  61 419.28 4.04 ****  62 409.14 4.22 ****  63 435.09 4.16 **** 64 435.12 4.27 ****  65 387.2 3.95 ****  66 414.17 4.24 ****  67 429.34.47 ****  68 359.19 3.89 ****  69 449.08 4.55 ****  70 375.25 4.19 **** 71 394.16 4.12 ****  72 403.15 4.49 ****  73 381.09 3.59 **** # 74  400.15 4.05 ****  75 387.22 4.29 ****  76 449.26 4.3 ****  77 391.284.19 ****  78 435.12 4.24 ****  79 437.19 4.49 ****  80 437.2 3.84 **** 81 375.03 3.57 ****  82 391.28 4.05 ****  83 425.28 4.16 ****  84359.22 3.95 ****  85 437.15 4.44 ****  86 399.19 4.22 ****  87 403.154.44 ****  88 399.19 4.17 ****  89 434.07 4.04 ****  90 387.23 4.26 **** 91 369.27 4.17 ****  92 377.29 4.04 ****  93 435.23 4.29 ****  94369.17 4.24 ****  95 449.06 4.51 ****  96 341.27 3.89 ****  97 387.194.2 ****  98 405.18 3.79 ****  99 469.22 4.29 **** 100 461.32 4.61 ****101 369.17 4.26 **** 102 413.28 4.02 **** 103 407.1 4.05 **** 104 375.274.11 **** 105 387.21 4.19 **** 106 373.18 4.04 **** 107 385.28 4.02 ****108 359.16 3.92 **** 109 369.34 4.16 **** 110 374.24 3.07 **** 111386.19 3.89 **** 112 369.27 2.63 **** 113 399.13 4.01 **** 114 389.34.05 **** 115 435.13 4.14 **** 116 407.16 4.09 **** 117 419.28 4.05 ****118 366.29 3.79 **** 119 521.19 4.16 **** 120 380.31 3.92 **** 121403.32 4.27 **** 122 383.31 4.37 **** 123 319.2 2.19 **** 124 351.142.53 *** 125 409.3 4.14 *** 126 423.3 3.95 *** 127 371.31 3.9 *** 128371.31 3.62 *** 129 449.13 3.81 *** 130 401.23 3.56 *** 131 385.22 3.74*** 132 363.06 2.31 *** 133 385.15 3.86 *** 134 377.3 4.04 *** 135397.15 2.42 *** 136 443.33 4.11 *** 137 361.07 2.53 *** 138 345.07 3.15*** 139 400.27 4.01 *** 140 488.23 4.36 *** 141 425.21 4.37 *** 142462.15 4.11 *** 143 369.23 3.74 *** 144 415.33 3.84 *** 145 361.3 4.39*** 146 400.21 3.81 *** 147 438.21 3.97 *** 148 469.01 4.42 *** 149425.25 4.24 *** 150 504.2 4.68 *** 151 397.01 2.44 *** 152 369.21 3.59*** 153 372.21 2.36 *** 154 377.29 3.97 *** 155 363.11 2.32 *** 156341.21 2.46 *** 157 407.14 1.78 *** 158 428.11 3.85 *** 159 351.13 2.47*** 160 450.15 3.95 *** 161 363.05 2.32 *** 162 325.26 2.66 *** 163319.2 2.24 *** 164 462.19 3.87 *** 165 371.31 3.65 *** 166 354.28 (-Boc)3.95 *** 167 432.16 3.87 *** 168 351.08 2.4 *** 169 385.35 4.09 *** 170351.07 2.51 *** 171 363.09 2.68 ** 172 384.21 3.52 ** 173 319.2 2.24 **174 N/A 2.38 ** 175 443.33 4.09 ** 176 417.30 2.77 ** 177 398.17 3.67 **178 363.11 2.31 ** 179 450.14 3.89 ** 180 421.19 2.65 ** 181 363.15 2.46** 182 419.14 4.14 ** 183 389.29 4.14 ** 184 431.27 4.1 ** 185 328.022.41 ** 186 462.19 3.81 ** 187 443.28 3.99 ** 188 446.19 3.81 ** 189405.19 3.8 ** 190 317.16 2.7 ** 191 369.23 3.89 ** 192 495.28 4.89 **193 297.2 2.53 ** 194 319.21 2.19 ** 195 494.25 2.79 ** 196 419.22 4.09** 197 317.16 2.41 ** 198 317.08 2.53 ** 199 448.24 3.95 ** 200 363.092.45 ** 201 365.09 2.36 ** 202 464.2 4.32 ** 203 301.18 2.27 ** 204429.23 3.57 ** 205 301.15 2.27 ** 206 476.3 4.33 ** 207 395.17 2.55 **208 367.36 2.72 ** 209 353.33 3.97 ** 210 313.21 2.33 ** 211 415.26 4.07** 212 389.2 2.88 ** 213 407.1 2.46 ** 214 357.07 2.48 ** 215 319.232.24 ** 216 283.1 2.41 ** 217 418.17 3.62 ** 218 435.23 3.77 ** 220308.23 2.37 ** 221 460.29 4.05 ** 222 365.11 2.52 ** 223 441.02 2.6 **224 341.27 2.6 ** 225 467.25 4.18 ** 226 369.34 4.01 ** 227 327.16 2.26** 228 369.34 2.64 ** 229 373.29 4.04 * 230 401.23 3.2 * 231 313.122.43 * 232 433.25 2.73 * 233 430.38 (-Boc) 4.34 * 234 351.17 2.4 * 235351.25 3.79 * 236 379.35 2.74 * 237 439.11 4.41 * 238 479.24 3.77 * 239328.16 2.35 * 240 307.27 3.87 * 241 523.19 3.7 * 242 438.27 4.14 * 243323.20 3.49 * 244 512 2.27 * 245 485 2.62 * 246 498 2.54 * 247 4712.36 * 248 283.23 2.24 * 249 339.17 3.07 * 250 355.30 3.57 * 251 297.262.26 * 252 341.21 2.44 * 253 301.27 2.29 * 254 301.25 2.27 * 255 281.312.2 * 256 345.2 2.26 * 257 335.21 2.34 * 258 459.27 3.72 * 259 479.243.52 * 260 287.26 2.36 * 261 287.26 2.56 * 262 380.24 3.92 * 263 503.503.20 * 264 369.36 2.52 * 265 355.26 2.54 * 266 355.26 2.42 * 267 370.223.61 * 268 355.26 2.42 * 269 355.27 2.37 * 270 370.23 3.19 * 271 369.342.62 * 272 374.31 2.90 * 273 492.25 2.76 * 274 451.30 3.17 * 275 374.312.61 * 276 374.31 2.72 * 277 349.28 1.5 * 278 457.28 4.11 * 279 *****280 407.10 3.92 * 281 508.15 4.74 * 282 507.08 4.42 * 283 422.32 3.86 *284 373.29 4.01 * 285 385.24 2.25 * 286 297.2 2.52 * 287 289.22 2.48 *288 461.26 2.57 * 289 380.29 3.82 * 290 396.27 3.60 * 291 299.17 2.43 *292 385.18 2.6 * 293 413.22 3.8 * 294 340.25 2.27 * 295 404.34 3.84 *296 299.17 2.23 * 297 326.24 2.4 * 298 235.13 2.18 * 299 351.16 2.62 *300 401 2.57 * 301 313.21 2.35 * 302 398.28 3.74 * 303 355.22 2.58 * 304440.32 4.09 * 305 341.08 2.48 * 306 364.3 3.65 * 307 350.32 3.35 * 308432.27 3.92 * 309 474.26 3.02 **** 310 289.03 2.35 * 311 345.19 2.58 *312 420.28 4.12 * 313 279.28 2.18 * 314 293.24 2.20 * 315 297.26 2.17 *316 472.26 3.85 * 317 428.25 3.95 * 318 309 2.25 * 319 284.09 2.1 * 320356.21 2.37 * 321 279.2 2.1 * 322 279.2 1.76 * 323 309.23 1.82 * 324280.19 1.76 * 325 279.2 1.76 * 326 263.17 1.93 * 327 343.18 2.33 * 328~0.005 4.16 * 329 0.0036 4.26 * 330 0.0047 4.24 * 331 ~0.010 2.94 *#332  ~0.010 4 * 333 410.27 3.64 ** 334 426.24 3.39 * 335 466.23 4.64*** 336 438.31 4.31 ** 337 454.24 4.63 *** 338 474.32 4.33 ** 339 412.33.83 * 340 446.33 4.49 * 341 447.26 4.25 *** 342 371.31 3.88 *** 343371.31 3.61 * 344 459.31 4.91 **** 345 383.35 4.44 **** 346 587 4.04**** 347 451.16 3.93 ***** 348 479.28 4.13 ***** 349 481.21 3.74 ****350 462.17 3.66 ***** 351 471.17 3.93 **** 352 403.29 3.98 **** 353497.16 3.94 ***** 354 525.2 4.19 ***** 355 511.21 3.81 ***** 356 490.33.93 ** 357 534.23 3.93 *** 358 433.2 3.45 *** 359 511.25 3.64 *** 360516 3.82 **** 361 474.26 3.02 **** 362 427 4.2 ***** 363 412.4 1.80 *364 484.3 2.49 ***** 365 457.3 4.06 *** 366 553.3 4.42 * 367 402.8 4.37**** 368 430.9 4.79 ** 369 427.0 4.06 ** 370 427.0 3.99 ***** 371 469.05.27 *** 372 486.9 4.96 * 373 470.8 5.01 *** 374 436.9 4.66 *** 375 4614.92 ** 376 385 3.79 ** 377 * 378 * 379 * 380 * 381 * 382 * 383 417.24.93 ***** 384 403.22 4.65 ***** 385 509.51 2.57 **** 386 465.26 2.52***** 387 465.26 2.52 ***** 388 495.4 3.94 ***** 389 538.3 4.29 *****390 480.5 3.23 ***** 391 562.55 3.63 ***** 392 443.4 3.88 ***** 393447.1 6.55 ***** 394 450.1 5.48 ***** 395 481.32 3.51 ***** 396 411.33.99 ***** 397 535.3 4.29 ***** 398 481.3 4.23 ***** 399 429.3 3.81***** 400 493.3 4.43 ***** 401 451.3 3.99 ***** 402 494.4 3.71 ***** 403479.3 4.23 ***** 404 473.6 3.78 ***** 405 551.17 4.58 ***** 406 425.44.13 ***** 407 457.4 4.04 ***** 408 425.4 4.09 ***** 409 477.4 4.18***** 410 451.3 3.99 ***** 411 443.4 3.86 ***** 412 473.4 4.23 ***** 413459.3 4.16 ***** 414 439.4 4.31 ***** 415 637.64 2.82 ***** 416 311.14.39 ***** 417 562.47 4.15 ***** 418 511.3 4.13 ***** 419 491.4 3.98***** 420 486.6 3.45 ***** 421 553.30 4.05 ***** 422 359.29 4.17 *****423 447.4 3.56 ***** 424 594.2 [M − H] 4.58 ***** 425 539.2 3.11 *****426 535.27 4.29 ***** 427 554.3 4.45 ***** 428 563.55 4.64 ***** 429564.42 2.77 ***** 430 431.3 3.41 ***** 431 522.2 5.05 ***** 432 489.44.14 ***** 433 578.44 2.82 ***** 434 467.18 4.11 ***** 435 444.3 3.95***** 436 477.4 3.93 ***** 437 543.4 3.92 ***** 438 500.1 4.35 ***** 439361.2 5.95 ***** 440 536.43 3.95 ***** 441 482.1 5.11 **** 442 367.12.92 **** 443 436.2 5.25 **** 444 455.28 3.73 **** 445 478 3.67 **** 446383.3 4.10 **** 447 464.9 5.11 **** 448 501.27 3.65 **** 449 482.24 2.62**** 450 587 4.04 **** 451 644.3 [M − H] 4.80 **** 452 439.3 3.56 ****453 553.1 6.13 **** 454 579.3 2.75 **** 455 583 3.84 **** 456 474.3 2.44**** 457 455 3.4 **** 458 456.3 2.51 **** 459 470.3 2.61 **** 460 509.304.16 **** 461 454.3 5.98 **** 462 580.56 2.85 **** 463 495.44 4.13 ****464 493.0 5.71 **** 465 507.4 3.98 **** 466 555.2 3.14 **** 467 524.24.02 **** 468 582.2 2.81 **** 469 525.2 5.07 **** 470 554.3 3.90 ****471 620.18 3.85 **** 472 335.3 5.52 **** 473 495.3 4.68 *** 474 511.24.99 *** 475 483 3.87 *** 476 400 3.45 *** 477 249.1 3.67 *** 478 525.13.25 *** 479 538.3 2.76 *** 480 456.1 4.26 *** 481 549.3 5.29 *** 482522.3 3.95 *** 483 470.1 4.46 *** 484 539.2 3.02 *** 485 398.9 4.18 ***486 349.1 6.03 *** 487 505 3.66 *** 488 555.2 3.34 *** 489 538.3 4.15*** 490 486.1 3.80 *** 491 537.31 2.64 *** 492 468 5.52 *** 493 504.32.68 *** 494 482.2 5.74 *** 495 403.3 4.16 *** 496 430.2 3.65 *** 497281.0 3.84 *** 498 481.4 4.81 *** 499 423.3 5.15 *** 500 506.29 3.85 ***501 534.3 2.68 *** 502 518.3 2.76 *** 503 508.2 5.72 *** 504 359.1 5.65*** 505 442.0 4.06 *** 506 386.3 5.32 *** 507 450 3.19 *** 508 397.15.97 *** 509 511.4 5.05 *** 510 321.4 5.19 *** 511 383.1 5.75 *** 512523.1 5.69 *** 513 361.1 5.12 *** 514 495.3 4.67 *** 515 363.5 6.34 **516 527.1 3.16 ** 517 464.2 5.86 ** 518 517.6 5.03 ** 519 527.2 3.88 **520 426.2 4.29 ** 521 509.4 4.99 ** 522 383.3 4.10 ** 523 439.0 6.11 **524 412.1 4.13 ** 525 4.95.3 3.46 ** 526 513.2 4.43 ** 527 535.3 4.94 **528 453.0 6.30 ** 529 481.3 3.43 ** 530 466.28 3.21 ** 531 549.6 5.21 **532 325.3 4.75 ** 533 506.2 4.96 ** 534 525.2 4.76 ** 535 541.2 3.51 **536 482.29 3.29 ** 537 476.3 2.51 ** 538 516.37 3.49 ** 539 337.3 [M −H] 2.14 ** 540 428.28 3.43 ** 541 525.2 4.42 ** 542 398.1 3.95 ** 543466.34 3.29 ** 544 723.58 3.92 ***** 545 466.31 3.28 ** 546 426.3 2.26** 547 335.2 5.45 ** 548 516.37 3.46 ** 549 414 2.89 ** 550 496 4.58 **551 544.5 2.78 ** 552 511.3 3.56 ** 553 440.9 5.75 ** 554 482.32 3.41 **555 372 2.89 ** 556 456.1 4.21 ** 557 538.4 3.71 ** 558 497.2 4.69 **559 460.8 4.96 ** 560 596.3 4.45 * 561 509.2 5.18 * 562 525.3 3.52 * 563483.1 4.96 * 564 432 2.18 * 565 276.9 4.00 * 566 384.4 1.73 * 567 511.44.71 * 568 295.0 4.14 * 569 480.21 3.50 ***** 570 549.22 4.59 ***** 571497.13 3.50 ** 572 525.29 4.14 ***** 573 341.34 2.14 **** 574 427.372.23 * 575 437.33 3.16 ** 576 575.43 3.71 *** 577 453.28 3.34 *** 578610.45 3.94 *** 579 481.32 3.51 ***** 580 495.29 3.64 ***** 581 465.433.64 * 582 516.34 3.31 * 583 512.26 3.39 *** 584 466.37 3.34 *** 585516.33 3.46 *** 586 387.27 2.13 ***** 587 467.29 3.66 *** 588 455.263.69 *** 589 471.3 3.83 *** 590 495.31 3.64 **** 591 541.35 3.73 *****592 523.42 3.58 ***** 593 541.38 3.69 **** 594 505.38 3.83 *** 595431.21 4.01 **** 596 431.24 3.99 ***** 597 445.24 4.19 ***** 598 459.244.36 ***** 599 513.17 4.19 **** 600 479.23 3.99 ***** 601 504.21 3.79**** 602 493.2 4.18 **** 603 513.16 4.19 **** 604 446.18 2.86 * 605503.23 3.84 ***** 606 461.19 3.46 *** 607 442.25 3.46 *** 608 489.2 3.72*** 609 433.27 3.98 ** 610 **** 611 ** 612 491.23 3.56 *** 613 513.144.18 **** 614 463 3.88 ** 615 381 3.48 *** 616 540 4.17 ** 617 621.574.13 **** 618 493.6 2.63 ***** 619 521.6 2.80 ***** 620 445.5 3.23 ****621 459.5 3.40 ***** 622 459.5 3.38 ***** 623 473.5 3.57 ***** 624 479.53.28 **** 625 507.6 3.53 ***** 626 493.6 3.48 **** 627 511.6 3.53 *****628 527.4 3.62 *** 629 527.5 3.72 ***** 630 573.5 3.75 ***** 631 507.63.65 ***** 632 538.6 3.53 **** 633 443.5 3.32 ***** 634 457.6 3.30 *****635 523.6 3.47 **** 636 463.6 3.12 ***** 637 621.62 2.77 ***** 638580.56 2.80 ***** 639 496.54 3.28 ***** 640 552.64 2.48 **** 641 445.554.13 ***** 642 381.49 3.97 ***** 643 397.47 3.95 ***** 644 395.45 3.78***** 645 521.15 4.17 ***** 646 531.11 4.58 **** 647 505.18 4.7 *****648 437.19 4.15 **** 649 477.21 4.1 ***** 650 487.18 4.3 **** 651 548.32.53 **** 652 419.23 4.15 **** 653 449.24 4.12 **** 654 433.26 4.3 *****655 453.19 4.33 **** 656 444.17 4.02 ***** 657 464.22 4.08 ***** 658461.6 4.30 ***** 659 489.7 4.78 ***** 660 543.7 4.92 ***** 661 459.53.63 ***** 662 471.5 3.87 ***** 663 491.6 3.63 ***** 664 507.6 3.80***** 665 485.6 3.85 **** 666 485.6 3.83 ***** 667 486.6 3.95 ***** 668503.6 3.58 ***** 669 521.6 3.88 ***** 670 521.6 4.02 ***** 671 501.64.13 ***** 672 501.6 4.10 ***** 673 539.6 4.02 674 555.6 4.13 **** 675555.6 4.22 **** 676 535.6 4.05 **** 677 535.6 4.15 **** 678 551.6 3.98*** 679 487.6 3.93 **** 680 599.5 4.27 ***** 681 566.6 4.02 **** 682496.5 2.13 ** 683 486.5 2.03 *** 684 484.6 2.67 *** 685 514.6 2.15 ***686 512.6 2.12 **** 687 510.6 2.13 *** 688 525.6 1.85 *** 689 494.5 3.12*** 690 524.6 2.32 *** 691 514.6 2.23 *** 692 512.6 2.35 *** 693 542.62.35 **** 694 540.6 2.27 **** 695 538.6 2.35 **** 696 553.6 2.07 *** 697522.6 3.95 ***** 698 578.5 2.43 **** 699 568.5 2.35 **** 700 566.6 2.45**** 701 596.6 2.47 **** 702 594.6 2.43 **** 703 592.6 2.48 **** 704607.6 2.20 *** 705 575.5 2.47 **** 706 576.5 3.58 ***** 707 477.51 2.77***** 708 491.53 2.73 ***** 709 503.55 2.68 ***** 710 495.45 4.42 *****711 475.51 4.62 ***** 712 513.50 4.42 ***** 713 529.46 4.62 **** 714509.51 4.43 ***** 715 482.46 4.28 ***** 716 457.47 4.05 **** 717 459.594.33 ***** 718 491.5 4.10 ***** 719 527.5 4.47 ***** 720 489.5 4.75***** 721 517.5 4.26 ***** 722 519.5 3.84 ***** 723 555.4 4.09 (nonpolar) ***** 724 541.54 2.90 ***** 725 478.47 3.58 ***** 726 516.5 2.67** 727 526.5 2.78 **** 728 544.5 2.80 *** 729 542.5 2.72 ***** 730 540.52.83 **** 731 555.6 2.43 *** 732 580.6 2.40 *** 733 523.5 2.78 ***** 734524.5 3.40 ***** 735 552.5 2.98 ***** 736 562.5 3.15 ***** 737 580.63.17 **** 738 578.5 3.02 ***** 739 576.6 3.17 ***** 740 591.6 2.75 ***741 616.5 2.62 *** 742 559.5 3.13 ***** 743 560.5 3.83 ***** 744 514.62.80 ***** 745 524.6 2.92 ***** 746 512.5 2.93 ***** 747 542.6 2.93***** 748 540.5 2.85 ***** 749 538.6 2.93 ***** 750 553.6 2.55 ***** 751521.5 2.92 **** 752 522.5 3.87 ***** 753 542.6 2.98 **** 754 552.6 *****755 540.6 3.17 **** 756 570.6 3.17 **** 757 568.6 3.07 ***** 758 566.63.17 *** 759 581.6 2.78 *** 760 549.6 3.13 ***** 761 550.5 4.17 *****762 544.5 2.68 **** 763 554.5 2.77 ***** 764 542.6 2.78 **** 765 572.52.75 **** 766 570.6 2.70 ***** 767 568.6 2.82 **** 768 583.6 2.47 ****769 608.6 2.38 *** 770 551.5 2.73 ***** 771 552.5 3.65 ***** 772 580.53.03 ***** 773 590.6 3.12 ***** 774 578.5 3.12 **** 775 608.6 3.05 *****776 606.5 3.05 ***** 777 604.6 3.12 ***** 778 619.6 2.77 ***** 779 644.52.63 *** 780 587.5 3.10 ***** 781 588.5 4.05 ***** 782 596.5 3.10 *****783 606.5 3.18 ***** 784 594.5 3.27 ***** 785 624.5 3.22 ***** 786 622.53.12 ***** 787 620.5 3.20 ***** 788 635.6 2.85 **** 789 660.5 2.68 ***790 603.5 3.22 ***** 791 604.5 4.25 ***** 792 480.50 2.98 ***** 793494.50 2.97 **** 794 494.50 2.97 *** 795 496.48 2.97 **** 796 563.502.41 **** 797 522.48 2.50 ***** 798 538.48 2.92 ***** 799 535.49 2.35*** 800 503.40 2.52 **** 801 504.43 3.42 ***** 802 504.42 3.37 ***** 803579.48 2.42 **** 804 538.48 2.43 ***** 805 584.50 2.52 ***** 806 554.402.47 ***** 807 540.47 2.50 ***** 808 551.48 2.33 **** 809 516.45 2.47***** 810 520.40 3.21 ***** 811 520.40 3.12 ***** 812 466.4 3.27 *****813 466.4 3.18 ***** 814 465.4 2.38 ***** 815 465.4 3.45 ***** # 816  497.4 2.70 ***** # 817   511.4 2.62 ***** # 818   491.4 2.43 **** 819494.4 3.53 ***** 820 494.4 3.47 **** 821 493.4 2.55 **** 822 493.4 3.73***** # 823   525.4 2.95 ***** # 824   539.4 2.83 ***** # 825   519.42.58 * 826 496.4 3.07 *** 827 496.4 2.98 **** 828 495.4 2.32 *** 829495.4 3.28 *** # 830   527.4 2.53 ***** # 831   541.4 2.50 ***** # 832  521.4 2.35 833 532.4 3.50 *** 834 532.4 3.42 **** 835 531.4 2.57 *** 836531.4 3.67 **** # 837   563.4 2.93 ***** # 838   577.4 2.82 ***** 839548.3 3.63 **** 840 548.3 3.58 **** # 841   579.3 3.08 ***** # 842  593.3 2.95 ***** # 843   573.4 2.75 ***** 844 451.91 3.58 *** 845 648.484.45 *** 846 526.45 2.57 *** 847 568.37 3.40 **** 848 585.30 3.57 *****849 604.37 3.52 **** 850 540.39 2.60 *** 851 495.06 4.37 ***** 852539.08 4.17 ***** 853 549.09 4.38 ***** 854 523.17 4.73 ***** 855 455.194.15 **** 856 495.18 4.10 ***** 857 505.16 4.30 ***** 858 566.3 2.57***** 859 437.22 4.15 ***** 860 467.2 4.13 ***** 861 451.12 4.10 ****862 471.17 4.32 ***** 863 514.55 4.38 ***** 864 462.28 4.00 **** 865482.13 4.08 **** 866 447.37 4.04 ***** 867 577.43 2.85 **** 868 477.144.37 ***** 869 504.53 3.62 ***** 870 493.55 2.80 ***** 871 489.54 2.72***** 872 493.55 2.80 ***** 873 503.54 2.73 ***** 874 479.2 2.74 *****875 425.52 4.27 ***** 876 492.52 3.57 ***** 877 489.54 2.72 ***** 878508.55 3.82 ***** 879 507.55 2.90 ***** 880 459.49 4.24 ***** 881 471.454.22 ***** 882 542.51 3.87 ***** 883 494.50 3.67 ***** 884 544.27 2.79***** 885 490.54 3.54 ***** 886 494.57 3.68 ***** 887 521.62 2.93 *****888 558.54 3.70 ***** 889 545.55 2.93 ***** 890 490.49 3.48 ***** 891528.49 3.69 ***** 892 546.50 3.75 ***** 893 461.49 4.36 ***** 894 580.472.72 ***** 895 491.51 2.77 ***** 896 576.49 4.00 ***** 897 504.51 3.52***** 898 457.53 4.25 ***** 899 481.37 4.17 ***** 900 541.55 3.00 *****901 575.54 2.98 ***** 902 471.49 4.12 ***** 903 621.39 2.72 ***** 904596.54 2.85 ***** 905 542.54 3.78 ***** 906 489.53 4.82 ***** 907 514.473.54 ***** 908 582.43 2.79 ***** 909 514.21 2.75 ***** 910 539.45 3.97***** 911 527.54 2.88 ***** 912 530.53 2.67 ***** 913 626.6 2.88 *****914 514.55 2.60 ***** 915 509.56 4.63 ***** 916 626.40 2.82 ***** 917561.46 2.95 ***** 918 642.56 2.85 ***** 919 543.45 4.82 ***** 920 557.572.87 ***** 921 527.39 4.52 ***** 922 561.53 2.85 ***** 923 612.51 2.92***** 924 498.20 2.71 ***** 925 596.54 2.88 ***** 926 5.62 3.85 *****927 540.65 4.25 ***** 928 510.52 3.10 ***** 929 506.46 2.95 ***** 930500.48 2.83 ***** 931 467.39 4.17 ***** 932 548.49 3.17 ***** 933 596.372.79 ***** 934 561.53 2.95 ***** 935 496.54 3.37 ***** 936 582.6 2.83***** 937 555.61 2.55 ***** 938 582.53 2.85 ***** 939 560.63 2.68 *****940 541.43 2.45 ***** 941 562.55 3.63 ***** 942 623.35 2.73 **** 943 4992.72 **** 944 525.56 4.36 **** 945 509.43 4.73 **** 946 566.53 2.77 ****947 510 2.44 **** 948 482.47 2.88 **** 949 524.55 3.22 **** 950 506.462.87 **** 951 544.53 3.27 **** 952 530.53 3.12 **** 953 552.46 2.90 ****954 403 4.11 **** 955 397 3.9 **** 956 484.55 2.42 **** 957 495.52 2.62**** 958 542.36 3.84 **** 959 496.24 2.81 **** 960 639.57 2.70 **** 961593.52 2.64 **** 962 516.59 2.65 **** 963 593.61 2.72 **** 964 598.552.83 **** 965 544.53 3.15 **** 966 564.45 3.32 **** 967 491.57 4.00 ****968 512.51 2.73 **** 969 492.46 2.90 **** 970 609.54 2.72 **** 971468.46 2.78 **** 972 496.47 3.02 **** 973 578.47 3.80 **** 974 528.343.79 *** 975 431.5 3.10 *** 976 564.46 3.23 *** 977 568.53 2.85 *** 978578.45 3.30 *** 979 470.55 2.45 *** 980 527.61 2.50 *** 981 560.51 3.12*** 982 425.60 3.78 *** 983 375.37 2.27 *** 984 5.06.19 3.97 ** 985407.31 1.82 * 986 531.56 2.17 * 987 497.1 4.4 ***** 988 605.62 2.52***** 989 564.61 2.55 ***** 990 610.62 2.67 ***** 991 580.58 2.60 ***992 566.61 2.60 *** 993 577.61 2.45 ***** 994 545.54 2.57 ***** 995546.57 3.53 ***** 996 578.46 3.71 ***** # (S) Isomer prepared andtested. Wherein: 1 star, >1 uM (1000 nM) 2 stars, 0.2 to 1 uM (200 nM to1000 nM) 3 stars, 0.04 uM to 0.2 uM (40 nM to 200 nM) 4 stars, 0.008 uMto 0.04 uM (8 nM to 40 nM) 5 stars, <0.008 uM (<8 nM)

Example 3 Compounds of the Invention Inhibit VEGF Expression and TumorGrowth, and Inhibit angiogenesis, in an In Vivo Tumor Growth PD Model

Compounds of the invention also show activity in the followingpharmacodynamic model that assesses tumor VEGF levels. Briefly, HT1080cells (a human fibrosarcoma cell line) may be implanted subcutaneouslyin nude mice. After seven days, mice may be administrated compoundsorally at a desired dosage range, e.g., 200 mg/kg/day, for seven days.The tumors may then be excised from mice, weighed and homogenized inTris-HCl buffer containing proteinase inhibitors. Moulder et al., CancerRes. 61(24):8887-95 (2001). Tumor VEGF levels are subsequently measuredusing a human VEGF ELISA kit (R&D System). Protein concentrations of thehomogenates are measured with a Bio-Rad Protein assay kit and tumor VEGFlevels are normalized to the protein concentrations.

Preferred compounds of the invention, when used for one week on a 100mm³ tumor, will generally inhibit tumor growth by at least 50%, ascompared to the vehicle-treated control groups. In similarly conductedexperiments, compounds of the invention are shown to reduce lung cancertumor growth (at a dosage of 3 mg/kg, twice a day, or 30 mg/kg, twice aday) (FIG. 2), Wilms and neuroblastoma tumor growth (at a dosage of 30mg/kg, once a day) (FIG. 3), fibrosarcoma tumor growth (at a dosage of10 mg/kg 3×/week) (FIG. 4), and human skin malignant melanoma (at adosage of 10 mg/kg 5×/week) (FIG. 5) as compared to vehicle.Furthermore, the compounds of the invention are shown to reduce VEGFplasma and tumor levels in fibrosarcoma tumors as compared to vehicle(at a dosage of 10 mg/kg 3×/week) (FIG. 6). The compounds of theinvention also inhibit tumor growth when administered together withother cancer agents, such as 5-FU (fluorouracil) in a colon cancer tumor(FIG. 7), taxol (paclitaxel) in a human cervical carcinoma (FIG. 8), andAvastin (bevacizumab) (FIG. 9). Furthermore, as shown in FIG. 10, thecompounds of the invention also reduce tumor and plasma VEGF levels whenadministered together with another cancer agent (such as Avastin).

The tumors excised from the mice can also be used in immunochemistryexperiments to determine levels of angiogenesis. As seen in FIG. 11,when antibodies to Collagen IV are used, angiogenesis is shown to beinhibited by the administration of a compound of the invention in Wilmstumor (SK-NEP-1) and neuroblastoma (SY5Y and NGP) tumors.

Example 4 Compounds of the Invention Do Not Affect VEGF mRNA Levels

Human solid tumor cells are treated with various concentrations of acompound of the present invention or a control under hypoxic conditions.Actinomycin D blocks transcription and is used as a control. Total RNAis isolated using the RNeasy kit (Qiagen, Inc., Valencia, Calif., USA)or an acid-guanidinium thiocyanate-phenol-chloroform based method(Chomczynski, P. and Sacchi, N., Anal. Biochem. 162: 156 (1987), herebyincorporated by reference).

Concentration of total RNA can be determined using the RiboGreen®fluorescent dye (Molecular Probes, Inc., Eugene, Oreg., USA) with theVersaFluor Fluorometer System (BioRad, Hercules, Calif., USA). Qualityand integrity of total RNA can be assessed on 1% formaldehyde-agarosegels. First-strand cDNA was synthesized using 1 μg of total RNA(DNase-treated) in a 20 μl reverse transcriptase reaction mixture(Leclerc G J, Barredo J C, Clin. Cancer Res. 2001, 7:942-951, which ishereby incorporated by reference). A region of the VEGF mRNA isamplified using PCR primers. The cDNA amplified fragment (approximately300 basepairs) is cloned into a vector such as the pCR2.1-TOPO vector(Invitrogen, Carlsbad, Calif., USA) to generate a plasmid. Serialten-fold dilutions (104 to 109 molecules) of this plasmid are used as areference molecule for the standard curve calculation (see Figure X).All Real-Time PCR reactions are performed in a 25 μl mixture containing1/20 volume of cDNA preparation (1 μl), 1× SYBR Green buffer (PE AppliedBiosystems, Foster City, Calif., USA), 4 mM MgCl2, 0.2 μM of each VEGFprimer used above, 0.2 mM dNTPs mix and 0.025 Unit of AmpliTaq Gold®thermostable DNA polymerase (Applied Biosystems, Foster City, Calif.,USA). Real-Time quantitations are performed using the BIO-RAD iCycler iQsystem (BioRad, Hercules, Calif., USA). The fluorescence threshold valuecan be calculated using the iCycle iQ system software. Using similarmethodologies, the data shown in FIG. 12 was obtained, and shows that acompound of the present invention does not affect VEGF mRNA levels

Example 5 Compounds of the Invention Do Not Affect the Activity of PDE5

The compounds of the invention are tested to assess their affect onphosphodiesterase 5 (PDE5) activity. The affect on PDE5 activity isdetermined using the High-Efficiency Fluorescence Polarization Assay(HEFP) kit from Molecular Devices. The HEFP assay measures the activityof PDE-5 by using fluorescein-labeled derivatives of cGMP as asubstrate. When hydrolyzed by PDE-5, fluorescein-labeled cGMPderivatives are able to bind to a binding reagent. The cGMPsubstrate:binding reagent complex results in a highly polarizedfluorescent state.

FIG. 13 shows the results of the compounds of the invention on PDE-5activity. After combining recombinant PDE5 (CalBioChem) and the cGMPsubstrate, the mixture is incubated at room temperature for 45 minutesin the presence or absence of compounds or a positive control(Tadalafil). The reaction is stopped upon addition of the bindingreagent. Fluorescence polarization is determined on a Viewlux using asetting recommended by the manufacturer. As is evident from FIG. 13, thecompounds of the invention do not inhibit the activity of PDE-5 incomparison to the positive control.

All publications and patent applications cited herein are incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

Although certain embodiments have been described in detail above, thosehaving ordinary skill in the art will clearly understand that manymodifications are possible in the embodiments without departing from theteachings thereof. All such modifications are intended to be encompassedwithin the claims of the invention.

1. A method for treating a solid tumor cancer comprising administering atherapeutically effective amount of a compound of formula:

or a pharmaceutically acceptable salt, racemate or stereoisomer of saidcompound, to a subject in need thereof; wherein: X is halogen; R_(o) ishalogen, substituted or unsubstituted C₁ to C₈ alkyl or OR_(a); R_(a) isH or C₁ to C₈ alkyl optionally substituted with one or more substituentsindependently selected from hydroxyl and halogen; and R_(d) is phenylsubstituted with one or more alkoxy or halogen substituents, wherein thecompound inhibits VEGF production in a HT1080 solid tumor grown in anude mouse, inhibits HT1080 solid tumor growth in a nude mouse orinhibits angiogenesis in a HT1080 solid tumor grown in a nude mouse. 2.The method of claim 1, wherein the compound has the formula:

or a pharmaceutically acceptable salt, racemate or stereoisomer thereof,wherein, X is halogen; R_(o) s OR_(a); R_(a) is H or C₁ to C₈ alkyloptionally substituted with one or more substituents independentlyselected from hydroxyl and halogen; and R_(d) is phenyl substituted withone or more alkoxy or halogen substituents.
 3. The method of claim 1,wherein the compound has the formula:

or a pharmaceutically acceptable salt, racemate or stereoisomer thereof,wherein, X is halogen; R_(a) is H or C₁ to C₈ alkyl optionallysubstituted with one or more substituents independently selected fromhydroxyl and halogen; and R_(d) is phenyl substituted with one or morehalogen substituents.
 4. The method of claim 1, wherein the compound hasthe formula:

or a pharmaceutically acceptable salt, racemate or stereoisomer thereof,wherein, X is halogen; R_(a) is C₁ to C₈ alkyl optionally substitutedwith one or more substituents independently selected from hydroxyl andhalogen; and R_(d) is phenyl substituted at the para position with ahalogen substituent.
 5. The method of claim 4, wherein X is chloro. 6.The method of claim 5, wherein R_(d) is phenyl substituted at the paraposition with chloro.
 7. The method of claim 6, wherein R_(a) isunsubstituted C₁ to C₈ alkyl.
 8. The method of claim 6, wherein R_(a) isC₁ to C₈ alkyl substituted with one or more hydroxyl substituents. 9.The method of claim 1 or 2, wherein said compound has a chiral carbon atthe point of attachment of the R_(o) substituted phenyl and saidcompound is an (S) isomer at said chiral carbon.
 10. The method of anyof claims 3-8, wherein said compound has a chiral carbon at the point ofattachment of the OR_(a) substituted phenyl and said compound is an (S)isomer at said chiral carbon.12.
 11. The method of claim 1, wherein thecompound has an EC₅₀ of less than 50 nM for inhibiting hypoxia-inducedVEGF expression in cultured HeLa cells.
 12. The method of claim 1,wherein the compound inhibits VEGF production in a HT1080 solid tumorgrown in a nude mouse.
 13. The method of claim 1, wherein the compoundinhibits HT1080 solid tumor growth in a nude mouse.
 14. The method ofclaim 1, wherein the compound inhibits angiogenesis in a HT1080 solidtumor grown in a nude mouse.
 15. A method for treating a solid tumorcancer comprising administering a therapeutically effective amount of acompound, wherein said compound is selected from the group consistingof:

or a pharmaceutically acceptable salt thereof, to a subject in needthereof, wherein the compound inhibits VEGF production in a HT1080 solidtumor grown in a nude mouse, inhibits HT1080 solid tumor growth in anude mouse or inhibits angiogenesis in a HT1080 solid tumor grown in anude mouse.
 16. The method of claim 15, wherein the compound has an EC₅₀of less than 50 nM for inhibiting hypoxia-induced VEGF expression incultured HeLa cells.
 17. The method of claim 15, wherein the compoundinhibits VEGF production in a HT1080 solid tumor grown in a nude mouse.18. The method of claim 15, wherein the compound inhibits HT1080 solidtumor growth in a nude mouse.
 19. The method of claim 15, wherein thecompound inhibits angiogenesis in a HT1080 solid tumor grown in a nudemouse.